U.S. patent application number 12/283482 was filed with the patent office on 2009-03-19 for methods of inhibiting hiv-2 infection.
Invention is credited to Thomas J. Ketas, William C. Olson.
Application Number | 20090074766 12/283482 |
Document ID | / |
Family ID | 40454723 |
Filed Date | 2009-03-19 |
United States Patent
Application |
20090074766 |
Kind Code |
A1 |
Ketas; Thomas J. ; et
al. |
March 19, 2009 |
Methods of inhibiting HIV-2 infection
Abstract
This invention provides methods of inhibiting HIV-2 infection of
a susceptible cell by HIV-2 which comprises subjecting the
susceptible cell to an effective HIV-2 infection inhibiting dose of
a humanized antibody designated PRO 140, or of an anti-CCR5
receptor monoclonal antibody, wherein the effective HIV-1 infection
inhibiting dose comprises from 0.1 mg per kg to 25 mg per kg of the
subject's body weight, so as to thereby inhibit the infection of
the susceptible cell by HIV-2.
Inventors: |
Ketas; Thomas J.; (Dumont,
NJ) ; Olson; William C.; (Yorktown Heights,
NY) |
Correspondence
Address: |
COOPER & DUNHAM, LLP
30 Rockefeller Plaza, 20th Floor
NEW YORK
NY
10112
US
|
Family ID: |
40454723 |
Appl. No.: |
12/283482 |
Filed: |
September 12, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60993804 |
Sep 14, 2007 |
|
|
|
Current U.S.
Class: |
424/133.1 ;
424/144.1 |
Current CPC
Class: |
A61K 2039/545 20130101;
A61K 39/3955 20130101; C07K 16/2866 20130101; A61K 39/3955
20130101; A61K 2300/00 20130101; A61K 2039/505 20130101; C07K
2317/24 20130101; A61K 45/06 20130101 |
Class at
Publication: |
424/133.1 ;
424/144.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395 |
Goverment Interests
[0001] This invention was made with support under United States
Government Grant No. AI066329 from the National Institute of
Allergy and Infectious Diseases. Accordingly, the United States
Government has certain rights in the subject invention.
Claims
1. A method of inhibiting HIV-2 infection of a susceptible cell by
HIV-2 in a subject which comprises subjecting the susceptible cell
to an effective HIV-2 infection inhibiting dose of (a) a humanized
antibody designated PRO 140, or of (b) an anti-CCR5 receptor
monoclonal antibody which (i) binds to CD4+CCR5+ cells and inhibits
fusion of HIV-1 with such cells, (ii) inhibits HIV-1 fusion with
CD4+CCR5+ cells with a potency equal or greater than that of PRO
140, (iii) coats CD4+CCR5+ cells in the subject without reducing
the number of such cells in the subject, and/or (iv) binds to the
subject's CD4+CCR5+ cells without inducing an increase in the
subject's plasma concentration of circulating .beta.-chemokines,
wherein PRO 140 comprises (i) two light chains, each light chain
comprising the light chain variable (V.sub.L) and constant
(C.sub.L) regions encoded by the plasmid designated pVK:HuPRO140-VK
(ATCC Deposit Designation PTA-4097), and (ii) two heavy chains,
each heavy chain comprising the heavy chain variable (V.sub.H) and
constant (C.sub.H) regions encoded either by the plasmid designated
pVg4:HuPRO140 HG2-VH (ATCC Deposit Designation PTA-4098) or by the
plasmid designated pVg4:HuPRO140 (mut B+D+I)-VH (ATCC Deposit
Designation PTA-4099), wherein the effective HIV-2 infection
inhibiting dose comprises from 0.1 mg per kg to 25 mg per kg of the
subject's body weight, so as to thereby inhibit the infection of
the susceptible cell by HIV-2.
2. The method of claim 1, wherein the susceptible cell is present
in a human subject.
3. The method of claim 1, wherein the anti-CCR5 receptor monoclonal
antibody binds to the same CCR5 epitope as that to which PRO 140
binds.
4. The method of claim 1, wherein the anti-CCR5 receptor monoclonal
antibody is a humanized, a human, or a chimeric antibody.
5. The method of claim 1, wherein the susceptible cell is subject
to an effective HIV-2 infection inhibiting dose of the antibody
designated PRO 140.
6. The method of claim 5, wherein the antibody designated PRO 140
comprises (i) two light chains, each light chain comprising the
light chain variable (V.sub.L) and constant (C.sub.L) regions
encoded by the plasmid designated pVK:HuPRO140-VK (ATCC Deposit
Designation PTA-4097) and (ii) two heavy chains, each heavy chain
comprising the heavy chain variable (V.sub.H) and constant
(C.sub.H) regions encoded by the plasmid designated pVg4:HuPRO140
HG2-VH (ATCC Deposit Designation PTA-4098).
7. The method of claim 1, wherein HIV-2 is of a subtype selected
from subtypes A, B, C, D, E, F, G, H, J, O, or a combination
thereof.
8. (canceled)
9. (canceled)
10. (canceled)
11. (canceled)
12. The method of claim 2, wherein the human subject is
administered the antibody designated PRO 140 which comprises (i)
two light chains, each light chain comprising the light chain
variable (V.sub.L) and constant (C.sub.L) regions encoded by the
plasmid designated pVK:HuPRO140-VK (ATCC Deposit Designation
PTA-4097) and (ii) two heavy chains, each heavy chain comprising
the heavy chain variable (V.sub.H) and constant (C.sub.H) regions
encoded by the plasmid designated pVg4:HuPRO140 HG2-VH (ATCC
Deposit Designation PTA-4098).
13. The method of claim 2, wherein the effective HIV-2 infection
inhibiting dose is (1) from 0.25 mg per kg to 25 mg per kg of the
subject's body weight; or (2) from 0.5 mg per kg to 25 mg per kg of
the subject's body weight; or (3) from 1 mg per kg to 5 mg per kg
of the subject's body weight.
14. (canceled)
15. (canceled)
16. The method of claim 2, wherein the effective HIV-2 infection
inhibiting dose is (1) 5 mg per kg of the subject's body weight; or
(2) 10 mg/kg of the subject's body weight; or (3) 20 mg/kg of the
subject's body weight.
17. (canceled)
18. (canceled)
19. The method of claim 2, wherein the effective HIV-2 infection
inhibiting dose is administered at regular intervals.
20. The method of claim 2, wherein the effective HIV-2 infection
inhibiting dose is sufficient to achieve in the subject a serum
concentration of the antibody of at least 400 ng/ml.
21. The method of claim 2, wherein the effective HIV-2 infection
inhibiting dose is sufficient to achieve and maintain in the
subject a serum concentration of the antibody of (1) at least 1
.mu.g/ml; or (2) about 3 to about 12 .mu.g/ml; or (3) at least 5
.mu.g/ml; or (4) at least 10 .mu.g/ml; or (5) at least 25 .mu.g/ml;
or (6) at least 50 .mu.g/ml.
22. (canceled)
23. (canceled)
24. (canceled)
25. (canceled)
26. (canceled)
27. The method of claim 2, wherein the effective HIV-2 infection
inhibiting dose is administered at one or more predefined
intervals.
28. (canceled)
29. (canceled)
30. (canceled)
31. (canceled)
32. (canceled)
33. (canceled)
34. (canceled)
35. (canceled)
36. (canceled)
37. The method of claim 2, wherein the antibody is administered to
the human subject via intravenous infusion or via subcutaneous
injection.
38. (canceled)
39. The method of claim 2, further comprising administering to the
human subject at least one additional antiretroviral agent
effective against HIV-2.
40. The method of claim 39, wherein the antiretroviral agent is a
CCR5 antagonist that does not compete with the humanized antibody
designated PRO 140 of (a), or the anti-CCR5 receptor monoclonal
antibody of (b).
41. The method of claim 40, wherein the CCR5 antagonist is an
antibody, a non-antibody, small-molecule CCR5 antagonist.
42. (canceled)
43. The method of claim 41, wherein the CCR5 antagonist is an
antibody which is a monoclonal antibody, humanized, a human, or a
chimeric antibody.
44. (canceled)
45. (canceled)
46. (canceled)
47. (canceled)
48. A method of (a) inhibiting in a human subject the onset or
progression of an HIV-2-associated disorder, or (b) of reducing the
likelihood of a human subject's contracting infection by HIV-2, the
inhibition of which or reduction being effected by inhibiting
fusion of HIV-2 to a susceptible cell in a subject, which comprises
administering to the subject at a predefined interval effective
fusion-inhibitory doses of a humanized antibody designated PRO 140,
or of an anti-CCR5 receptor antibody which (i) binds to CD4+CCR5+
cells and inhibits fusion of HIV-1 with such cells, (ii) inhibits
HIV-1 fusion with CD4+CCR5+ cells with a potency equal or greater
than that of PRO-140 (iii) coats CD4+CCR5+ cells in the subject
without reducing the number of such cells in the subject, and/or
(iv) binds to the subject's CD4+CCR5+ cells without inducing an
increase in the subject's plasma concentration of circulating
.beta.-chemokines, wherein PRO 140 comprises (i) two light chains,
each light chain comprising the light chain variable (V.sub.L) and
constant (C.sub.L) regions encoded by the plasmid designated
pVK:HuPRO140-VK (ATCC Deposit Designation PTA-4097), and (ii) two
heavy chains, each heavy chain comprising the heavy chain variable
(V.sub.H) and constant (C.sub.H) regions encoded either by the
plasmid designated pVg4:HuPRO140 HG2-VH (ATCC Deposit Designation
PTA-4098) or by the plasmid designated pVg4:HuPRO140 (mut B+D+I)-VH
(ATCC Deposit Designation PTA-4099), wherein each administration of
the antibody delivers to the subject from 0.1 mg per kg to 25 mg
per kg of the subject's body weight, so as to thereby inhibit the
onset or progression of the HIV-2-associated disorder in the
subject or reduce the likelihood of a human subject's contracting
infection by HIV-2.
49. (canceled)
50. (canceled)
51. (canceled)
Description
[0002] Throughout this application, various publications are
referenced in parentheses by author name and date, or by a patent
or patent publication number. Full citations for these publications
may be found at the end of the specification immediately preceding
the claims. The disclosures of each of these publications in its
entirety are hereby incorporated by reference into this application
in order to more fully describe the state of the art as known to
those skilled therein as of the date of this application.
BACKGROUND OF THE INVENTION
[0003] In 1984, 3 years after the first reports of a disease that
was to become known as AIDS, researchers discovered the primary
causative viral agent, the human immunodeficiency virus type 1
(HIV-1). Human immunodeficiency virus type 2 (HIV-2) was identified
in West Africa in 1986 (Clavel et al. 1986 Isolation of a new human
retrovirus from West African patients with AIDS Science
233:343-346) The similarity of HIV-2 to HIV-1 has been
investigated. HIV-1 and HIV-2 share genetic and biological
properties, such as genome structure, mechanisms for
transactivation, and CD4 cell depletion (Guyader et al. 1987 Genome
organization and transactivation of the human immunodeficiency
virus type 2 Nature 326:662-669) HIV-1 and HIV-2 have the same mode
of transmission and are associated with similar opportunistic
infections and AIDS. In persons infected with HIV-2,
immunodeficiency seems to develop more slowly and to be milder.
Compared with persons infected with HIV-1, those with HIV-2 are
less infectious early in the course of infection due to a longer
clinical latency period (Ancelle et al. 1987 Long incubation period
of HIV-2 infection Lancet i:688-689). As the disease advances,
HIV-2 infectiousness seems to increase; however, compared with
HIV-1, the duration of this increased infectiousness is shorter and
the viral load in the asymptomatic stage is lower (Simon et al.
1993 Cellular and plasma viral load in patients infected with HIV-2
AIDS 7:1411-1417). HIV-1 and HIV-2 also differ in geographic
patterns of infection, which are predominantly found in Africa.
[0004] Infection of cells by HIV-1 or HIV-2 is mediated by the
viral envelope (Env) glycoproteins gp120 and gp41, which are
expressed as a noncovalent, oligomeric complex on the surface of
virus and virally infected cells. Entry of the virus into target
cells proceeds through a cascade of events at the cell surface that
include (1) binding of the viral surface glycoprotein gp120 to a
cell surface receptor, (2) Env binding to fusion coreceptors, and
(3) multiple conformational changes in gp41. The first
high-affinity interaction between the virion and the cell surface
is the binding of gp120 to cell surface CD4, which is the primary
receptor for HIV-1 (Dalgleish et al.; 1984; Klatzmann et al., 1984;
Maddon et al., 1986; McDougal et al., 1986). This binding induces
conformational changes in gp120, which enable it to interact with
one of several chemokine receptors (Berger, 1997; Bieniasz et al.,
1998; Dragic et al., 1997; Littman, 1998). The CC-chemokine
receptor 5 (CCR5) is the major co-receptor for macrophage-tropic
(R5) strains, and plays a crucial role in the transmission of HIV-1
(Berger, 1997; Bieniasz et al., 1998; Dragic et al., 1997; Littman,
1998). T cell line-tropic (X4) viruses use CXCR4 to enter target
cells, and usually, but not always, emerge late in disease
progression or as a consequence of virus propagation in tissue
culture. Some primary HIV-1 isolates are dual-tropic (R5X4) since
they can use both co-receptors, though not always with the same
efficiency (Connor et al., 1997; Simmons et al., 1996). Binding of
gp120 to a chemokine receptor in turn triggers conformational
changes in the viral transmembrane glycoprotein gp41, which
mediates fusion of the viral and cellular membranes. Each stage of
this multi-step process can be blocked with inhibitors of the
appropriate viral or cellular protein, and the inhibitors of gp120,
gp41, CD4 and coreceptor are collectively known as entry
inhibitors. Entry inhibitors represent at least 4 distinct classes
of agents based on their molecular targets and determinants of
viral resistance (Olson and Maddon, 2003). Table 1 lists HIV-1
entry inhibitors known to be in clinical development or approved
for clinical use.
[0005] PRO 542 is a tetravalent, third-generation CD4-IgG2 fusion
protein comprising the D1D2 domains of CD4 genetically fused to the
heavy and light chain constant regions of human IgG2 (Allaway et
al., 1995; Zhu et al., 2001). This agent binds the HIV-1 envelope
glycoprotein gp120 with nanomolar affinity and may inhibit virus
attachment both by receptor blockade and by detaching gp120 from
the virion surface, thereby irreversibly inactivating the
virus.
TABLE-US-00001 TABLE 1 HIV-1 entry inhibitors Compound Molecular
Class Target Stage of Entry Developer PRO542 CD4-Ig Fusion Protein
gp120 Attachment Progenics BMS-488043 Small Molecule gp120
Attachment Bristol-Myers Squibb TNX-355 Humanized antibody CD4
Post-Attachment Tanox PRO 140 Humanized antibody CCR5 Coreceptor
Progenics CCR5mAb004 Human antibody CCR5 Coreceptor Human Genome
Sciences SCH-D Small Molecule CCR5 Coreceptor Schering-Plough
(vicriviroc) UK-427,857 Small Molecule CCR5 Coreceptor Pfizer
(maraviroc) GW873140 Small Molecule CCR5 Coreceptor GlaxoSmithKline
TAK-652 Small Molecule CCR5 Coreceptor Takeda AMD070 Small Molecule
CXCR4 Coreceptor AnorMed T- Peptide gp41 gp41 Fusion Trimeris/Roche
20 (enfuvirtide) BMS-488043 is an optimized analog of BMS-378806
(see PCT International Publication Nos. WO 01/62255 A1 and WO
03/082289 A1), which has been variously reported to block gp120
attachment to CD4 (Lin et al., 2002; 2003) and post-attachment
events (Si et al., 2004). TNX-355 is a humanized IgG4 version of
the anti-CD4 monoclonal antibody (mAb) 5A8, which blocks fusion
events that occur post-attachment of gp120 to CD4 (Burkly et al.,
1992; Moore et al., 1992). PRO 140, a humanized anti-CCR5 mAb, and
the small-molecule CCR5 antagonists, SCH-D (also now designated SCH
417670 or vicriviroc), UK-427,857 (also designated maraviroc) and
GW873140, are discussed below. CCR5mAb004 is a fully human mAb,
generated using the Abgenix XenoMouse .RTM. technology, that
specifically recognizes and binds to CCR5 (Roschke et al., 2004).
CCR5mAb004 has been reported to inhibit CCR5-dependent entry of
HIV-1 viruses into human cells, and recently entered Phase 1
clinical trials (HGS Press Release, 2005). The first small-molecule
anti-CCR5 antagonist identified as capable of inhibiting HIV-I
infection was TAK-779 (Baba et al., 1999). However, TAK-779
exhibited poor oral bioavailability (Baba et al., 2005) and local
injection site irritation (Iizawa et al., 2003), and has been
replaced in clinical development by a TAK-779 derivative, TAK-652
(Baba et al., 2005). TAK-652 is an orally bioavailable CCR5
antagonist with potent anti-HIV-1 activity in the nanomolar range
in vitro and promising pharmacological profiles in vivo (Baba et
al., 2005). AMD070 is a second-generation CXCR4 inhibitor; the
first-generation CXCR4 inhibitor AMD3100 did not demonstrate a
favorable safety window for HIV-1 therapy (Schols et al., 2002).
Finally, T-20 was approved for salvage therapy of HIV-1 infection
following favorable antiviral and safety profiles in each of two
pivotal Phase 3 studies (Lalezari et al., 2003; Lazzarin et al.,
2003).
CCR5 as a Target for Anti-HIV-1 Therapy
[0006] As first demonstrated in 1986, HIV-1 binds to target cells
via the CD4 receptor but requires additional host cell factors to
mediate entry (Maddon et al., 1986). Over the next decade, a number
of candidate coreceptors were proposed, but none reproducibly
mediated viral entry when coexpressed with CD4 in otherwise
nonpermissive cells. However, in 1996, certain chemokine receptors,
mainly CCR5 and CXCR4, were shown to serve as requisite fusion
coreceptors for HIV-1.
[0007] Cocchi et al. (1995) provided the first link between HIV-1
and chemokines, which are small (.about.8 kDa) homologous soluble
proteins. Chemokines mediate the recruitment and activation of
immune cells. They are classified as CC-, CXC-, CX.sub.3C- and
XC-chemokines based on the number and sequential relationship of
the first two of four conserved cysteine residues; most are either
CC- or CXC-chemokines. The CC-chemokines RANTES, MIP-1.alpha. and
MIP-1.beta., were shown to block replication of primary
macrophage-tropic strains of HIV-1 (Cocchi et al., 1995). Using
expression cloning techniques, Feng et al. (1996) discovered that
the chemokine receptor fusin (later renamed CXCR4) was a fusion
coreceptor for strains of HIV-1 adapted to growth on T cell lines.
Shortly thereafter, several groups reported the cloning of CCR5, a
CC chemokine receptor with specificity for RANTES, MIP-1.alpha. and
MIP-1.beta. (Combadiere et al., 1996; Raport et al., 1996; Samson
et al., 1997), and others then demonstrated that CCR5 was the main
entry cofactor used by primary macrophage-tropic HIV-1 isolates
(Alkhatib et al., 1996; Choe et al., 1996; Deng et al., 1996;
Doranz et al., 1996; Dragic et al., 1996). The patterns of CCR5 and
CXCR4 expression helped solve long-standing riddles concerning the
tropism of different strains of HIV-1. Macrophage-tropic,
T-cell-line-tropic and dual-tropic viruses could be more
descriptively classified as being R5, X4 and R5X4 viruses based on
their abilities to utilize CCR5, CXCR4 or both receptors,
respectively, for entry.
[0008] A variety of other chemokine receptors can function as HIV-1
coreceptors when over-expressed in vitro. The list includes CCR8,
Apj, V28, US28, CCR2b, CCR3, gprl, Bonzo (STRL33, TYMSTR), and BOB
(gprl5). Clearly, proteins belonging to the chemokine receptor
family have biochemical properties that promote HIV-1 membrane
fusion. However, most of the above-mentioned coreceptors are not
very efficient, are not normally coexpressed with CD4, and function
only with certain strains of HIV-1, HIV-2 or SIV. The in vivo
relevance of these alternative coreceptors has not been
established.
[0009] Several factors make CCR5 an attractive target for new
antiretroviral therapies. CCR5 plays a central role in HIV-1
transmission and pathogenesis, and naturally-occurring mutations in
CCR5 confer protection from HIV-1 infection and disease
progression. The most notable CCR5 polymorphism involves a 32 bp
deletion in the coding region of CCR5 (A32) (Liu et al., 1996). The
A32 allele encodes a nonfunctional receptor that fails to reach the
cell surface. Individuals who possess one normal and one mutant
CCR5 gene express lower levels of CCR5, and their T cells are less
susceptible to R5 virus infection in vitro (Liu et al., 1996; Wu et
al., 1997). A32 heterozygotes experience a milder course of disease
characterized by reduced viral burdens and delayed progression to
AIDS (Huang et al., 1996; Michael et al., 1997). These results
support the concept that reducing CCR5 availability can lower viral
replication and slow disease progression. Individuals with two
mutant CCR5 genes comprise a significant fraction of people of
northern European descent; the demography is suggestive of a prior
pandemic of a CCR5-using pathogen. Such individuals represent human
CCR5 "knockouts" in that they do not express a functional CCR5
protein. Except in rare instances (Balotta et al., 1997; Biti et
al., 1997; O'Brien et al., 1997), these individuals are resistant
to HIV-1 infection (Huang et al., 1996; Liu et al., 1996; Michael
et al., 1997; Samson et al., 1997), and their T cells cannot be
infected with R5 viruses in vitro (Liu et al., 1996). These
findings underscore the central role of CCR5 in HIV-1 transmission.
In fact, it is now known that R5 viruses mediate transmission in
nearly all cases and mediate progression to AIDS in most cases.
[0010] Importantly, individuals who lack CCR5 enjoy normal health
and display no obvious immunologic or other defects. This may
reflect the redundancy of chemokine signaling pathways and the
rather limited pattern of expression of CCR5. CCR5 expression is
largely confined to activated T cells and macrophages, which
represent the primary targets for HIV-1 infection in vivo, although
low-level CCR5 expression has been reported on other tissues, such
as smooth muscle (Schecter et al., 2000).
[0011] CCR5 knockout mice have been generated and provide further
insight into the effects of abrogating CCR5 function. CCR5 knockout
mice develop normally and are ostensibly healthy, although minor
alterations in immune responses can be observed upon challenge with
particular pathogens (Huffnagle et al., 1999; Schuh et al., 2002;
Tran et al., 2000; Zhou et al., 1998). In contrast, the CXCR4
knockout is a lethal phenotype in mice (Lapidot et al., 2001), and
has not been observed in humans.
[0012] Taken together, these genetic analyses strongly support a
new therapeutic approach based on CCR5 as a drug target. The
error-prone nature of reverse transcriptase generates immense
genetic diversity that fosters the development of drug-resistant
isolates, and HIV-1's ability to utilize multiple fusion
coreceptors provides one path to resistance. Drug-resistant viruses
have been isolated for all marketed antiretrovirals, which
nevertheless provide important therapeutic benefit when used in
appropriate combinations. Thus, despite the potential emergence of
drug-resistant viruses, CCR5-targeting agents may serve as a new
treatment paradigm for HIV-1 infection.
[0013] Although the apparent non-essential nature of CCR5 suggests
that CCR5 antagonists may be well tolerated in vivo, further
studies are required to determine that long-term effects of
abrogating CCR5 function in individuals whose immune systems
developed in its presence. Such potentially deleterious effects may
be mitigated by use of agents that bind to CCR5 and inhibit binding
of HIV-1 thereto, but do not impair normal CCR5 function. One agent
demonstrated to have such properties is the humanized anti-CCR5
mAb, PRO 140, which effectively blocks HIV-1 replication at
concentrations that do not inhibit the physiologic activity of CCR5
(Olson et al., 1999). PRO 140 was identified using a fluorescence
resonance energy transfer (RET) assay screen for anti-HIV activity.
It is potently antiviral, having an IC.sub.90 of about 4 .mu.g/ml
(Olson et al., 1999; Trkola et al., 2001) and protects diverse
primary target cell types (Ketas et al., 2003; Olson and Maddon,
2003). Repeated administration of PRO 140 led to prolonged control
of HIV-1 replication without viral escape in the hu-PBL SCID mouse
model, and PRO 140 is currently in Phase 1 human clinical
trials.
[0014] Subsequent to the identification of the small-molecule CCR5
antagonist, TAK-779 (Baba et al., 1999), several other
small-molecule CCR5 antagonists have been identified. Four of these
(SCH-C, SCH-D, UK-427,857, GW873140) have completed similarly
designed Phase 1 studies in HIV-infected individuals (Reynes et
al., 2002; Schurmann et al., 2004; Dorr et al., 2003; Lalezari et
al., 2004). Each of these agents mediated dose-dependent .about.1
log.sub.10 mean reductions in HIV-1 RNA levels during the treatment
period of 10-14 days. As expected, viral loads rebounded to
baseline levels following cessation of therapy. The most common
drug-related side-effects were neurologic (headache, dizziness) and
gastrointestinal (nausea, diarrhea, flatulence), and these were not
dose limiting. With the exception of SCH-C (Reyes et al., 2001),
none of the above-identified agents induced clinically significant
changes in QTc intervals.
[0015] A double-blind, placebo-controlled, single oral dose study
has also been conducted to evaluate the safety, tolerability, and
pharmacokinetics of TAK-652, the successor compound to TAK-779, in
healthy male volunteers (Baba et al., 2005). The single
administration of TAK-652 solution was reportedly safe and well
tolerated (Baba et al., 2005).
[0016] Overall, these studies provide preliminary validation of
CCR5 as a target for HIV-1 therapy. While the small-molecule CCR5
antagonists represent patentably distinct chemical series with
differing pharmacokinetic and metabolic properties, the compounds
share many properties in their inhibition of CCR5 function, binding
site on CCR5, resistance profiles, and dosing regimen. These
similarities may conceivably limit the number of genuine treatment
options afforded by small-molecule CCR5 antagonists. Moreover, it
remains to be determined whether there are untoward consequences of
chronic blockade of CCR5 function, and the utility of
small-molecule CCR5 antagonists for HIV-1 therapy remains to be
established by demonstration of appropriate safety and efficacy in
Phase 3 clinical studies.
Monoclonal Antibody Therapeutics
[0017] In recent years, mAb products have provided new standards of
care in diverse disease settings. Currently, 18 mAbs are approved
by the U.S. Food and Drug Administration (FDA) for indications
including cancer, autoimmune disease, transplant rejection and
viral infection. Notably, 14 mAbs have been approved since 2000. In
many instances, mAbs provide safety, efficacy and ease-of-use
profiles that are unrivalled by small-molecule compounds. Examples
include Synagis (MedImmune, Inc., Gaithersburg, Md.), a humanized
mAb to respiratory syncytial virus (RSV), and Rituxan (Genentech,
San Francisco, Calif.), an anti-CD20 mAb that provides the standard
of care for non-Hodgkin's lymphoma.
[0018] The humanized anti-CCR5 mAb, PRO 140, is structurally,
functionally and mechanistically distinct from the small-molecule
CCR5 antagonists and therefore represents a unique CCR5 inhibitor
class. PRO 140 is a humanized version of the murine mAb, PA14,
which was generated against CD4.sup.+CCR5.sup.+ cells (Olson et
al., 1999). PRO 140 binds to CCR5 expressed on the surface of a
cell, and potently inhibits HIV-1 entry and replication at
concentrations that do not affect CCR5 chemokine receptor activity
in vitro and in the hu-PBL-SCID mouse model of HIV-1 infection
(Olson et al., 1999; Trkola et al., 2001). The latter finding
provides in vivo proof-of-concept for PRO 140 anti-HIV-1 therapy,
and PRO 140 is currently undergoing Phase 1a clinical studies.
[0019] Important differences between PRO 140 and small-molecule
CCR5 antagonists are summarized in Table 2. It is evident from
Table 2 that, whereas small-molecule CCR5 antagonists in
development share many properties, PRO 140 is clearly distinct from
these small-molecule inhibitors. The differences between the two
CCR5 inhibitor classes reveal that PRO 140 may offer a
fundamentally distinct, and in many ways complementary, product
profile from that of small-molecule CCR5 antagonists. Indeed, PRO
140 represents a novel therapeutic approach to treating HIV-1
infection and could play an important role in HIV-1 therapy
irrespective of whether small-molecule CCR5 antagonists are
ultimately clinically approved.
Synergistic Inhibition of HIV-1 Infection by Different Classes of
Inhibitors
[0020] Synergistic inhibition of HIV-1 entry has previously been
demonstrated using certain anti-Env antibodies in combination with
other anti-Env antibodies (Thali et al., 1992; Tilley et al., 1992;
Laal et al., 1994; Vijh-Warrier et al., 1996; Li et al., 1997; Li
et al., 1998), anti-CD4 antibodies (Burkly et al., 1995), or
CD4-based proteins (Allaway et al., 1993). Similarly, synergies
have been observed using anti-CCR5 antibodies in combination with
other anti-CCR5 antibodies, CC-chemokines, or CD4-based proteins
(Olson et al., 1999). Prior studies described in PCT International
Publication No. WO 00/35409, published Jun. 22, 2000, examined
combinations of HIV-1 attachment inhibitors and CCR5 coreceptor
inhibitors. Prior studies described in PCT International
Publication No. WO 01/55439, published Aug. 2, 2001, examined
combinations of inhibitors of gp41 fusion intermediates and HIV-1
attachment. Prior studies described in PCT International
Publication No. WO 02/22077, published Mar. 21, 2002, examined
combinations of fusion inhibitors and CCR5 coreceptor inhibitors,
as well as the triple combination of fusion inhibitors, CCR5
coreceptor inhibitors and HIV-1 attachment inhibitors. However, no
prior study has examined the combination of different classes of
CCR5 coreceptor inhibitors, such as anti-CCR5 mAbs and non-antibody
CCR5 antagonists.
TABLE-US-00002 TABLE 2 Comparison of PRO 140 and small-molecule
CCR5 antagonists under development Small Molecules PRO 140
Identification Screen Chemokine Binding HIV-1 Entry Block Natural
Activity of CCR5 Yes No Potential for Immune Yes No
Suppression/Dysregulation Tolerability Cardiac, Neurological No
Toxicity Toxicities for some Binding site on CCR5 Common
Hydrophobic Extracellular Epitope that Pocket defined by spans
Multiple Hydrophilic Transmembrane Regions of Domains CCR5 Viral
Cross-Resistance Significant Limited Development of Resistance In
Vitro 6 to 19 weeks None at 40 weeks Drug-Drug Interactions
Significant Unlikely Food Interactions Significant Unlikely Dosing
Once or Twice Daily Biweekly to Monthly
SUMMARY OF THE INVENTION
[0021] This invention provides a method of inhibiting HIV-2
infection of a susceptible cell by HIV-2 which comprises subjecting
the susceptible cell to an effective HIV-2 infection inhibiting
dose of (a) a humanized antibody designated PRO 140, or of (b) an
anti-CCR5 receptor monoclonal antibody which (i) binds to CD4+CCR5+
cells and inhibits fusion of HIV-1 with such cells, (ii) inhibits
HIV-1 fusion with CD4+CCR5+ cells with a potency equal or greater
than that of PRO 140, (iii) coats CD4+CCR5+ cells in the subject
without reducing the number of such cells in the subject, and/or
(iv) binds to the subject's CD4+CCR5+ cells without inducing an
increase in the subject's plasma concentration of circulating
.beta.-chemokines, wherein PRO 140 comprises (i) two light chains,
each light chain comprising the light chain variable (V.sub.L) and
constant (C.sub.L) regions encoded by the plasmid designated
pVK:HuPRO140-VK (ATCC Deposit Designation PTA-4097), and (ii) two
heavy chains, each heavy chain comprising the heavy chain variable
(V.sub.H) and constant (C.sub.H) regions encoded either by the
plasmid designated pVg4:HuPRO140 HG2-VH (ATCC Deposit Designation
PTA4098) or by the plasmid designated pVg4:HuPRO140 (mut B+D+I)-VH
(ATCC Deposit Designation PTA-4099), wherein the effective HIV-2
infection inhibiting dose comprises from 0.1 mg per kg to 25 mg per
kg of the subject's body weight, so as to thereby inhibit the
infection of the susceptible cell by HIV-2.
[0022] This invention also provides a method of inhibiting HIV-2
infection in an HIV-2-infected human subject which comprises
administering to the subject an effective HIV-2 infection
inhibiting dose of (a) a humanized antibody designated PRO 140, or
of (b) an anti-CCR5 receptor monoclonal antibody which (i) binds to
CD4+CCR5+ cells in the subject and inhibits fusion of HIV-1 with
such cells, (ii) inhibits HIV-1 fusion with CD4+CCR5+ cells with a
potency equal or greater than that of PRO 140, (iii) coats
CD4+CCR5+ cells in the subject without reducing the number of such
cells in the subject, and/or (iv) binds to the subject's CD4+CCR5+
cells without inducing an increase in the subject's plasma
concentration of circulating .beta.-chemokines, wherein PRO 140
comprises (i) two light chains, each light chain comprising the
light chain variable (V.sub.L) and constant (C.sub.L) regions
encoded by the plasmid designated pVK:HuPRO140-VK (ATCC Deposit
Designation PTA4097), and (ii) two heavy chains, each heavy chain
comprising the heavy chain variable (V.sub.H) and constant
(C.sub.H) regions encoded either by the plasmid designated
pVg4:HuPRO140 HG2-VH (ATCC Deposit Designation PTA4098) or by the
plasmid designated pVg4:HuPRO140 (mut B+D+I)-VH (ATCC Deposit
Designation PTA-4099), wherein the effective HIV-2 infection
inhibiting dose comprises from 0.1 mg per kg to 25 mg per kg of the
subject's body weight, so as to thereby inhibit HIV-2 infection in
the subject.
[0023] This invention also provides a method of inhibiting in a
human subject the onset or progression of an HIV-2-associated
disorder, the inhibition of which is effected by inhibiting fusion
of HIV-2 which comprises administering to the subject at a
predefined interval effective fusion-inhibitory doses of a
humanized antibody designated PRO 140, or of an anti-CCR5 receptor
antibody which (i) binds to CD4+CCR5+ cells in the subject and
inhibits fusion of HIV-1 with such cells, (ii) inhibits HIV-1
fusion with the subject's CD4+CCR5+ cells with a potency
characterized by an IC90 of 10 .mu.g/ml or less, (iii) coats the
subject's CD4+CCR5+ cells without reducing the number of such cells
in the subject, and/or (iv) binds to the subject's CD4+CCR5+ cells
without inducing an increase in the subject's plasma concentration
of circulating .beta.-chemokines, wherein PRO 140 comprises (i) two
light chains, each light chain comprising the light chain variable
(V.sub.L) and constant (C.sub.L) regions encoded by the plasmid
designated pVK:HuPRO140-VK (ATCC Deposit Designation PTA4097), and
(ii) two heavy chains, each heavy chain comprising the heavy chain
variable (V.sub.H) and constant (C.sub.H) regions encoded either by
the plasmid designated pVg4:HuPRO140 HG2-VH (ATCC Deposit
Designation PTA-4098) or by the plasmid designated pVg4:HuPRO140
(mut B+D+I)-VH (ATCC Deposit Designation PTA-4099), wherein each
administration of the antibody delivers to the subject from 0.1 mg
per kg to 25 mg per kg of the subject's body weight, so as to
thereby inhibit the onset or progression of the HIV-2-associated
disorder in the subject.
[0024] This invention further provides a method of reducing the
likelihood of a human subject's contracting infection by HIV-2
which comprises administering to the subject at a predefined
interval effective fusion-inhibitory doses of a humanized antibody
designated PRO 140, or of an anti-CCR5 receptor antibody which (i)
binds to CD4+CCR5+ cells in the subject and inhibits fusion of
HIV-1 with such cells, (ii) inhibits HIV-1 fusion with the
subject's CD4+CCR5+ cells with a potency characterized by an IC90
of 10 .mu.g/ml or less, (iii) coats the subject's CD4+CCR5+ cells
without reducing the number of such cells in the subject, and/or
(iv) binds to the subject's CD4+CCR5+ cells without inducing an
increase in the subject's plasma concentration of circulating
.beta.-chemokines, wherein PRO 140 comprises (i) two light chains,
each light chain comprising the light chain variable (V.sub.L) and
constant (C.sub.L) regions encoded by the plasmid designated
pVK:HuPRO140-VK (ATCC Deposit Designation PTA4097), and (ii) two
heavy chains, each heavy chain comprising the heavy chain variable
(V.sub.H) and constant (C.sub.H) regions encoded either by the
plasmid designated pVg4:HuPRO140 HG2-VH (ATCC Deposit Designation
PTA-4098) or by the plasmid designated pVg4:HuPRO140 (mut B+D+I)-VH
(ATCC Deposit Designation PTA4099), wherein each administration of
the antibody delivers to the subject from 0.1 mg per kg to 25 mg
per kg of the subject's body weight, so as to thereby reduce the
likelihood of the subject's contracting an HIV-2 infection.
BRIEF DESCRIPTION OF THE FIGURES
[0025] FIG. 1
[0026] Humanized PRO 140 is potently antiviral. The in vitro
neutralization activity of murine and humanized PRO 140 was tested
against four primary R5 HIV-1 isolates using a whole virus
replication assay. The data reflect the median values from 8 or
more independent assays. The genetic subtypes of the viruses are
indicated in parentheses.
[0027] FIG. 2
[0028] Antiviral activity is independent of target cell. Inhibition
of infection of four different target cells by three primary R5
HIV-1 isolates with was tested.
[0029] FIG. 3
[0030] In vitro HIV-1 susceptibility to PRO 140 quantified using
the PhenoSense.TM. entry assay. PRO 140 was tested for activity
against 20 primary HIV-1 isolates in the PhenoSense HIV Entry.TM.
assay at ViroLogic, Inc. (now Monogram Biosciences, South San
Francisco, Calif.). Drug susceptibility is reported as IC.sub.50
values, which represent the concentration required for 50%
inhibition of viral infectivity.
[0031] FIG. 4
[0032] PRO 140 blocks HIV-1 but not chemokine signaling. The
effects of PRO 140 on the inhibition of RANTES-induced calcium
mobilization in L1.2-CCR5 cells and on inhibition of
HIV-1.sub.JR-FL replication in PBMC cultures were determined.
Similar results were obtained for MIP-1.alpha. and MIP-1.beta..
[0033] FIG. 5
[0034] PRO 140 provides prolonged control of viral replication in
HIV-1-infected mice. SCID mice were reconstituted with normal human
peripheral blood mononuclear cells and infected 2 weeks later with
HIV-1.sub.JR-CSF. Multiple doses of PRO 140 were administered
following attainment of steady state viral levels. Plasma viral
loads pre- and post-injection are indicated.
[0035] FIG. 6
[0036] PRO 140 coats but does not deplete CCR5 lymphocytes. Healthy
male volunteers (n=4) were treated with a single intravenous
infusion of PRO 140 at a dose level of 2 mg/kg. At the indicated
times post-treatment, blood was collected and analyzed for CCR5
lymphocyte levels. The group mean values and standard deviations
are indicated.
[0037] FIG. 7
[0038] Serum concentrations of PRO 140. Healthy male volunteers
were treated with a single intravenous infusion of PRO 140 at dose
levels of 0.1, 0.5 and 2.0 mg/kg, as indicated. At the indicated
times post-treatment, serum was collected, cryopreserved, and
analyzed for PRO 140 levels. Data for individual patients are
indicated.
[0039] FIG. 8
[0040] PRO 140 does not affect plasma chemokine levels. Healthy
male volunteers were treated with a single intravenous infusion of
0.1 mg/kg PRO 140 (Cohort 1), 0.5 mg/kg PRO 140 (Cohort 2) or
matched placebo. At the indicated times post-treatment, plasma was
collected, cryopreserved and analyzed for levels of RANTES. The
Lower Limit of Quantification of the assay was 415 pg RANTES/mL
plasma. Data represent the group mean values.
[0041] FIG. 9
[0042] Synergistic inhibition of HIV-1 fusion exhibited by PRO 140
with different compounds. Interactions between PRO 140 and
small-molecule, peptide, mAb, and chimeric CD4-immunoglobulin
inhibitors of CCR5, CD4, gp120 and gp41 targets for inhibiting
HIV-1 fusion were assessed using the RET assay. Mean combination
index (CI) values with 95% confidence intervals are plotted for
data obtained using the compounds combined in a 1:1 molar ratio. A
CI value of <1 indicates synergistic interactions; a CI value of
1 indicates additive interactions; and a CI value of >1
indicates antagonistic interactions.
[0043] FIG. 10
[0044] PRO 140 coats but does not deplete lymphocytes. Healthy male
volunteers (n=4) were treated with a single intravenous infusion of
PRO 140 at a dose level of 5 mg/kg. At the indicated times
post-treatment, blood was collected and analyzed for CCR5
lymphocyte levels. The group mean values and standard deviations
are indicated.
[0045] FIG. 11
[0046] Dose-response curves for inhibition of HIV-1.sub.JR-FL
envelope-mediated membrane fusion by combinations of CCR5
inhibitors. Dilutions were analyzed in triplicate wells, and the
data points depict the mean and standard deviations of replicates.
(A) PRO 140 and UK-427,857 were tested individually and in a 1:1
fixed molar ratio over the indicated range of concentrations. In
the experiment depicted, IC50 and IC90 values were 2.9 nM and 11 nM
for PRO 140, 5.0 nM and 21 nM for UK-427,857, and 2.1 nM and 4.6 nM
for the combination. CI50 and CI90 values were 0.58 and 0.32,
respectively. (B) SCH-D and UK-427,857 were tested individually and
in a 1:1 fixed molar ratio over the indicated range of
concentrations. In the experiment depicted, IC50 and IC90 values
were 5.5 nM and 34 nM for SCH-D, 9.7 nM and 59 nM for UK-427,857,
and 6.1 nM and 31 nM for the combination. CI50 and CI90 values were
0.87 and 0.73, respectively.
[0047] FIG. 12
[0048] Inhibition of PRO 140-PE binding to CEM.NKR-CCR5 cells by
unlabeled PRO 140, UK-427,857 and SCH-D. CEM.NKR-CCR5 cells were
incubated with varying concentrations of unlabeled PRO 140,
UK-427,857 or SCH-D for 30 min at room temperature in PBSA buffer
prior to addition of 5 nM PRO 140-PE for an additional 30 min.
Cells were washed and then analyzed by flow cytometry for both the
mean fluorescence intensity (MFI) of binding and the percent of
cells gated for positive binding of PRO 140-PE. Inhibition was
assessed on the basis of both MFI (A) and percent cells gated
(B).
[0049] FIG. 13
[0050] Inhibition of .sup.3H-UK-427,857 binding by unlabeled
UK-427,857, SCH-D and PRO 140. (A) CEM.NKR-CCR5 cells were
pre-incubated with varying concentrations of unlabeled UK-427,857,
SCH-D or PRO 140 for 30 min in PBSA buffer at ambient temperature
prior to the addition of at 2 nM .sup.3H-UK-427,857 for an
additional 30 min. Cells were washed and then analyzed for
radioactivity by scintillation counting. (B) The stability of
UK-427,857 binding under the assay conditions was examined by
pre-incubating CEM.NKR-CCR5 cells with 2 nM .sup.3H-UK-427,857
prior to washing, addition of unlabeled compounds for 30 min, and
processing as described above.
[0051] FIG. 14
[0052] Table showing results of Phase 1b Study.
[0053] FIG. 15
[0054] Graph depicting change in viral load from baseline in Phase
1b Study.
[0055] FIGS. 16A and 16B:
[0056] Graphs depicting change in CD4+ cell counts in Phase 1b
Study.
[0057] FIG. 17: Graph depicting mean log.sub.10 in HIV-1 RNA change
from baseline in Phase 1b Study.
[0058] FIG. 18: Graph depicting mean log.sub.10 change in HIV RNA,
day 10 results and individual subject nadirs.
[0059] FIG. 19: Graph depicting virological response rate
determined at the completion of the study. Percent of subjects in
study cohorts with .gtoreq.1 log.sub.10 reduction in HIV-1 RNA.
[0060] FIG. 20: Coreceptor virus tropism (Trofile.TM., Monogram
Biosciences).
[0061] FIG. 21: Median PRO 140 and PA14 EC.sub.50 (ug/ml) values
for HIV-2 isolates. Each of the five HIV-2 R5 isolates was
susceptible to inhibition by both PRO 140 and PA14. Neither PRO 140
nor PA14 had any measurable effect on replication of R5X4 HIV-2
isolates. EC.sub.50 values for HIV-1 were similar to those for
HIV-2 (0.09 to 0.46 ug/ml for HIV-1; 0.07 to 7.6 ug/ml for HIV-2).
The EC.sub.50 values observed for PRO 140 and PA14 were not
significantly different in this study (p=0.11, two-tailed Wilcoxon
signed rank test). The median maximum percent inhibition (MPI)
values were >94% for each of the R5 HIV-1 and HIV-2 isolates
tested in this study.
[0062] FIGS. 22A, 22B, and 22C: Inhibition data were analyzed using
GraphPad Prism software (Graphpad Software, San Diego, Calif.)
[0063] A. Graph depicting results of PRO 140 vs R5 HIV-1 inhibition
assay. B. Graph depicting results of PRO 140 vs R5 HIV-2 inhibition
assay. C. Graph depicting results of PRO 140 vs R5X4 HIV-2
inhibition assay.
DETAILED DESCRIPTION OF THE INVENTION
[0064] As used in this application, except as otherwise expressly
provided herein, each of the following terms shall have the meaning
set forth below.
[0065] "Administering" refers to delivering in a manner which is
effected or performed using any of the various methods and delivery
systems known to those skilled in the art. Administering can be
performed, for example, topically, intravascularly, intravenously,
pericardially, orally, parenterally, via implant, transmucosally,
dermally, transdermally, intradermally, intramuscularly,
subcutaneously, intraperitoneally, intrathecally,
intralymphatically, intralesionally, epidurally, rectally,
intravaginally, intraocularly, intrasinally, nasally,
intraspinally, mucosally, transmucosally, transplacentally or by in
vivo electroporation. An agent or composition may also be
administered in an aerosol, such as for pulmonary and/or intranasal
delivery. Administering can also be performed, for example, once, a
plurality of times, and/or over one or more extended periods.
[0066] An "antibody" shall include, without limitation, an
immunoglobulin molecule comprising two heavy chains and two light
chains and which recognizes an antigen. The immunoglobulin molecule
may derive from any of the commonly known classes, including but
not limited to IgA, secretory IgA, IgG and IgM. IgG subclasses are
also well known to those in the art and include but are not limited
to human IgG1, IgG2, IgG3 and IgG4. "Antibody" includes, by way of
example, both naturally occurring and non-naturally occurring
antibodies; monoclonal and polyclonal antibodies; chimeric and
humanized antibodies; human or nonhuman antibodies; wholly
synthetic antibodies; and single chain antibodies. A nonhuman
antibody may be humanized by recombinant methods to reduce its
immunogenicity in man. Methods for humanizing antibodies are well
known to those skilled in the art. "Antibody" also includes,
without limitation, a fragment or portion of any of the
afore-mentioned immunoglobulin molecules and includes a monovalent
and a divalent fragment or portion. Antibody fragments include, for
example, Fc fragments and antigen-binding fragments (Fab).
[0067] An "anti-chemokine receptor antibody" refers to an antibody
which recognizes and binds to an epitope on a chemokine receptor.
As used herein, "anti-CCR5 antibody" refers to an antibody which
recognizes and binds to an epitope on the CCR5 chemokine
receptor.
[0068] "Attachment" means the process that is mediated by the
binding of the HIV-1 envelope glycoprotein to the human CD4
receptor, which is not a fusion coreceptor.
[0069] As used herein, "CCR5" or "R5", is a chemokine receptor
which binds members of the C--C group of chemokines and whose amino
acid sequence comprises that provided in Genbank Accession Number
1705896 and related polymorphic variants. As used herein, CCR5
includes, without limitation, extracellular portions of CCR5
capable of binding the HIV-1 envelope protein. "CCR5" and "CCR5
receptor" are used synonymously.
[0070] "CD4" means the mature, native, membrane-bound CD4 protein
comprising a cytoplasmic domain, a hydrophobic transmembrane
domain, and an extracellular domain which binds to the HIV-1 gp120
envelope glycoprotein.
[0071] "CDR", or complementarity determining region, means a highly
variable sequence of amino acids in the variable domain of an
antibody.
[0072] A "cell" includes a biological cell, e.g., a HeLa cell, a
lymphocyte, a PBMN cell, and a non-biological cell, e.g., a
phospholipid vesicle or virion. A "cell susceptible to HIV
infection" may also be referred to as a "target cell" and includes
a cell capable of being infected by or fusing with HIV or an
HIV-infected cell.
[0073] "CXCR4" or "R4" is a chemokine receptor which binds members
of the C--X--C group of chemokines and whose amino acid sequence
comprises that provided in Genbank Accession No 400654 and related
polymorphic variants. As used herein, CXCR4 includes extracellular
portions of CXCR4 capable of binding the HIV-1 envelope
protein.
[0074] "Exposed" to HIV-1 refers to contact with HIV-1 such that
infection could result.
[0075] A "fully human" antibody refers to an antibody wherein all
of the amino acids correspond to amino acids in human
immunoglobulin molecules. "Fully human" and "human" are used
synonymously.
[0076] "HIV" refers to the human immunodeficiency virus. HIV shall
include, without limitation, HIV-1. HIV-1 includes but is not
limited to extracellular virus particles and the forms of HIV-1
associated with HIV-1 infected cells. The human immunodeficiency
virus (HIV) may be either of the two known types of HIV (HIV-1 or
HIV-2). The HIV-1 virus may represent any of the known major
subtypes (classes A, B, C, D, E, F, G, H, or J), outlying subtype
(Group 0), or an as yet to be determined subtype of HIV-1.
HIV-1.sub.JR-FL is a strain that was originally isolated at autopsy
from the brain tissue of an AIDS patient. The virus has been cloned
and the DNA sequences of its envelope glycoproteins are known
(GenBank Accession No. U63632). In terms of sensitivity to
inhibitors of viral entry, HIV-1.sub.JR-FL is known to be highly
representative of primary HIV-1 isolates. "JRCSF" refers to a HIV-1
isolate of subtype B. JRCSF is a strain originally isolated from
cerebral spinal fluid and brain tissue of an AIDS patient (Science
236, 819-822, 1987). The virus has been cloned and its genome DNA
sequence is known (GenBank Accession No. M38429). Unlike HIV
isolate JRFL, JRCSF does not productively infect macrophages.
[0077] A "humanized" antibody refers to an antibody wherein some,
most or all of the amino acids outside the CDR regions are replaced
with corresponding amino acids derived from human immunoglobulin
molecules. In one embodiment of the humanized forms of the
antibodies, some, most or all of the amino acids outside the CDR
regions have been replaced with amino acids from human
immunoglobulin molecules, whereas some, most or all amino acids
within one or more CDR regions are unchanged. Small additions,
deletions, insertions, substitutions or modifications of amino
acids are permissible as long as they do not abrogate the ability
of the antibody to bind a given antigen. Suitable human
immunoglobulin molecules include IgG1, IgG2, IgG3, IgG4, IgA, IgE
and IgM molecules. A "humanized" antibody retains an antigenic
specificity similar to that of the original antibody.
[0078] One skilled in the art would know how to make the humanized
antibodies of the subject invention. Various publications, several
of which are hereby incorporated by reference into this
application, also describe how to make humanized antibodies. For
example, the methods described in U.S. Pat. No. 4,816,567 comprise
the production of chimeric antibodies having a variable region of
one antibody and a constant region of another antibody.
[0079] U.S. Pat. No. 5,225,539 describes another approach for the
production of a humanized antibody. This patent describes the use
of recombinant DNA technology to produce a humanized antibody
wherein the CDRs of a variable region of one immunoglobulin are
replaced with the CDRs from an immunoglobulin with a different
specificity such that the humanized antibody would recognize the
desired target but would not be recognized in a significant way by
the human subject's immune system. Specifically, site-directed
mutagenesis is used to graft the CDRs onto the framework. Other
approaches for humanizing an antibody are described in U.S. Pat.
Nos. 5,585,089 and 5,693,761, and PCT International Publication No.
WO 90/07861, which describe methods for producing humanized
immunoglobulins. These have one or more CDRs and possible
additional amino acids from a donor immunoglobulin and a framework
region from an accepting human immunoglobulin. These patents
describe a method to increase the affinity of an antibody for the
desired antigen. Some amino acids in the framework are chosen to be
the same as the amino acids at those positions in the donor rather
than in the acceptor. Specifically, these patents describe the
preparation of a humanized antibody that binds to a receptor by
combining the CDRs of a mouse monoclonal antibody with human
immunoglobulin framework and constant regions. Human framework
regions can be chosen to maximize homology with the mouse sequence.
A computer model can be used to identify amino acids in the
framework region which are likely to interact with the CDRs or the
specific antigen and then mouse amino acids can be used at these
positions to create the humanized antibody. The above methods are
merely illustrative of some of the methods that one skilled in the
art could employ to make humanized antibodies. Methods for making
fully human antibodies are also well known to one skilled in the
art. For example, fully human monoclonal antibodies can be prepared
by immunizing animals transgenic for large portions of human
immunoglobulin heavy and light chain loci. See, e.g., U.S. Pat.
Nos. 5,591,669, 5,545,806, 5,545,807, 6,150,584, and references
cited therein, the contents of which are incorporated herein by
reference. These transgenic animals have been genetically modified
such that there is a functional deletion in the production of
endogenous (e.g., murine) antibodies. The animals are further
modified to contain all or a portion of the human germ-line
immunoglobulin gene locus such that immunization of these animals
will result in the production of fully human antibodies to the
antigen of interest. Following immunization of these animals (e.g.,
XenoMouse.RTM. (Abgenix), HuMAb-Mouse.RTM. (Medarex/GenPharm)),
monoclonal antibodies can be prepared according to standard
hybridoma technology. These monoclonal antibodies will have human
immunoglobulin amino acid sequences and therefore will not provoke
human anti-mouse antibody (HAMA) responses when administered to
humans. In vitro methods also exist for producing human antibodies.
These include phage display technology (U.S. Pat. Nos. 5,565,332
and 5,573,905) and in vitro stimulation of human B cells (U.S. Pat.
Nos. 5,229,275 and 5,567,610). The contents of these patents are
incorporated herein by reference.
[0080] Nucleic acids encoding heavy and light chains of the
humanized PRO 140 antibody have been deposited with the ATCC.
Specifically, the plasmids designated pVK-HuPRO140, pVg4-HuPRO140
(mut B+D+I) and pVg4-HuPRO140 HG2, respectively, were deposited
pursuant to, and in satisfaction of, the requirements of the
Budapest Treaty with the ATCC, Manassas, Va., U.S.A. 20108, on Feb.
22, 2002, under ATCC Accession Nos. PTA 4097, PTA 4099 and PTA
4098, respectively.
[0081] The half-life of the humanized PRO 140 antibody may be
increased to prolong exposure of the drug following administration.
For example, the half-life of PRO 140 in serum or plasma may be
extended, and/or the amount and time that PRO 140 coats CCR5+
target cells may be extended. Illustrative methods include
conjugation to polyethylene glycol (PEG), (pegylation), or
monomethoxypolyethylene glycol (mPEG); and molecularly engineering
PRO 140, e.g., by site directed mutagenesis, to have altered
pH-dependent binding to the human neonatal Fc receptor (FcRn), an
MHC class I-like Fc receptor. (See, e.g., S. B. Petkova et al.,
2006, Int'l Immunol., 18(12):1759-1769; P. R. Hinton et al., 2006,
J. Immunol., 176:346-356).
[0082] The production of antibody or antibody fragment-polymer
conjugates having an effective size or molecular weight that
confers an increase in serum half-life, an increase in mean
residence time in circulation (MRT) and/or a decrease in serum
clearance rate over underivatized antibody or antibody fragments.
The antibody fragment-polymer conjugates can be made by
derivatizing the desired antibody fragment with an inert polymer.
It will be appreciated that any inert polymer which provides the
conjugate with the desired apparent size, or which has the selected
actual molecular weight, is suitable for use in constructing
suitable antibody fragment-polymer conjugates. Many inert polymers
are suitable for use in pharmaceuticals. See, e.g., Davis et al.,
Biomedical Polymers: Polymeric Materials and Pharmaceuticals for
Biomedical Use, pp. 441-451 (1980). A non-proteinaceous polymer is
particularly advantageous. The non-proteinaceous polymer ordinarily
is a hydrophilic synthetic polymer, i.e., a polymer not otherwise
found in nature. However, polymers which exist in nature and are
produced by recombinant or in vitro methods are also useful, as are
polymers which are isolated from native sources. Hydrophilic
polyvinyl polymers, e.g., polyvinylalcohol and
polyvinvypyrrolidone, are suitable. Particularly useful are
polyalkylene ethers such as polyethylene glycol (PEG);
polyoxyalklyenes such as polyoxyethylene, polyoxypropylene and
block copolymers of polyoxyethylene and polyoxypropylene
(Pluronics); polymethacrylates; carbomers; branched or unbranched
polysaccharides which comprise the saccharide monomers D-mannose,
D- and L-galactose, fucose, fructose, D-xylose, L-arabinose,
D-glucuronic acid, sialic acid, D-galacturonic acid, D-mannuronic
acid (e.g., polymannuronic acid, or alginic acid), D-glucosamine,
D-galactosamine, D-glucose and neuraminic acid including
homopolysaccharides and heteropolysaccharides such as lactose,
amylopectin, starch, hydroxyethyl starch, amylose, dextran sulfate,
dextran, dextrins, glycogen, or the polysaccharide subunit of acid
mucopolysaccharides, e.g., hyaluronic acid, polymers of sugar
alcohols such as polysorbitol and polymannitol, heparin or heparon.
The polymer prior to cross-linking need not, but can be, water
soluble, but the final conjugate needs to be water soluble. The
conjugate exhibits a water solubility of at least about 0.01 mg/ml,
or at least about 0.1 mg/ml, or at least about 1 mg/ml. In addition
the polymer should not be highly immunogenic in the conjugate form,
nor should it possess viscosity that is incompatible with
intravenous infusion or injection if the conjugate is intended to
be administered by such routes. In one embodiment, the polymer
contains only a single group that is reactive. This helps to avoid
cross-linking of protein molecules. However, reaction conditions
can be maximized to reduce cross-linking, or to purify the reaction
products through gel filtration or ion-exchange chromatography to
recover substantially homogeneous derivatives. In other
embodiments, the polymer contains two or more reactive groups for
the purpose of linking multiple antibody fragments to the polymer
backbone. Again, gel filtration or ion-exchange chromatography can
be used to recover the desired derivative in substantially
homogeneous form. The molecular weight of the polymer can range up
to about 500,000 Daltons (D) and can be at least about 20,000 D, or
at least about 30,000 D, or at least about 40,000 D. The molecular
weight chosen can depend upon the effective size of the conjugate
to be achieved, the nature (e.g., structure such as linear or
branched) of the polymer and the degree of derivitization, i.e.,
the number of polymer molecules per antibody fragment, and the
polymer attachment site or sites on the antibody fragment. The
polymer can be covalently linked to the antibody or fragment
thereof through a multifunctional crosslinking agent, which reacts
with the polymer and one or more amino acid residues of the
antibody or fragment to be linked. The polymer may be crosslinked
directly by reacting a derivatized polymer with the antibody or
antibody fragment, or vice versa. The covalent crosslinking site on
the antibody or antibody fragment includes the N-terminal amino
group and epsilon amino groups found on lysine residues, as well
other amino, imino, carboxyl, sulfhydryl, hydroxyl, or other
hydrophilic groups. The polymer may be covalently bonded directly
to the antibody or antibody fragment without the use of a
multifunctional (ordinarily bifunctional) crosslinking agent, as
described, for example, in U.S. Pat. No. 6,458,355. The degree of
substitution with such a polymer will vary depending upon the
number of reactive sites on the antibody or fragment thereof, the
molecular weight, hydrophilicity and other characteristics of the
polymer, and the particular antibody or antibody fragment
derivitization sites chosen. In general, the conjugate contains
from 1 to about 10 polymer molecules, but greater numbers of
polymer molecules attached to the antibodies or antibody fragments
are also contemplated. The desired amount of derivitization is
easily achieved by using an experimental matrix in which the time,
temperature and other reaction conditions are varied to change the
degree of substitution, after which the level of polymer
substitution of the conjugates is determined by size exclusion
chromatography or other means known and practiced in the art.
Functionalized polyethylene glycol (PEG) polymers to modify the
antibody or antibody fragments are available from Shearwater
Polymers, Inc. (Huntsville, Ala.). Such commercially available PEG
derivatives include, but are not limited to, amino-PEG, PEG amino
acid esters, PEG-hydrazide, PEG-thiol, PEG-succinate,
carboxymethylated PEG, PEG-propionic acid, PEG amino acids, PEG
succinimidyl succinate, PEG succinimidyl propionate, succinimidyl
ester of carboxymethylated PEG, succinimidyl carbonate of PEG,
succinimidyl esters of amino acid PEGs, PEG-oxycarbonylimidazole,
PEG-nitrophenyl carbonate, PEG tresylate, PEG-glycidyl ether,
PEG-aldehyde, PEG-vinylsulfone, PEG-maleimide,
PEG-orthopyridyl-disulfide, heterofunctional PEGs, PEG vinyl
derivatives, PEG silanes and PEG phospholides. The reaction
conditions for coupling these PEG derivatives will vary depending
on the protein, the desired degree of PEGylation and the PEG
derivative utilized. Some factors involved in the choice of PEG
derivatives include: the desired point of attachment (such as
lysine or cysteine R-groups), hydrolytic stability and reactivity
of the derivatives, stability, toxicity and antigenicity of the
linkage, suitability for analysis, etc. Specific instructions for
the use of any particular derivative are available from the
manufacturer. The resulting conjugates are separated from the
unreacted starting materials by gel filtration or ion exchange
HPLC.
[0083] "Monoclonal antibodies," also designated a mAbs, are
antibody molecules whose primary sequences are essentially
identical and which exhibit the same antigenic specificity.
Monoclonal antibodies may be produced by hybridoma, recombinant,
transgenic or other techniques known to those skilled in the
art.
[0084] A "non-antibody antagonist of a CCR5 receptor" (or "R5")
refers to an agent that does not comprise an antibody, and which
binds to a CCR5 receptor and inhibits the activity of this
receptor. Such inhibition can include inhibiting the binding of
HIV-1 to the CCR5 receptor. By way of example, non-antibody
antagonists include nucleic acids, carbohydrates, lipids,
oligopeptides, non-chemokines and non-protein, small organic
molecules.
[0085] A "small-molecule" CCR5 receptor antagonist includes, for
example, a small organic molecule, or a non-protein small organic
molecule, which binds to a CCR5 receptor and inhibits the activity
of the receptor. Such inhibition includes, e.g., inhibiting the
binding of HIV-1 to the receptor or inhibiting the entry of HIV-1
into a susceptible cell. In one embodiment, the small organic
molecule has a molecular weight less than 1,500 daltons. In another
embodiment, the molecule has a molecular weight less than 600
daltons.
[0086] "Subject" includes any animal or artificially modified
animal capable of becoming infected with HIV. Animals include, but
are not limited to, humans, non-human primates, dogs, cats,
rabbits, ferrets, and rodents such as mice, rats and guinea pigs.
Artificially modified animals include, but are not limited to, SCID
mice with human immune systems. In an embodiment, the subject is a
human. In an embodiment, the subject is a human patient.
[0087] "Synergy" between two or more agents refers to the combined
effect of the agents which is greater than their additive effects.
Illustratively, agents may be peptides, proteins, such as
antibodies, small molecules, organic compounds, and drug forms
thereof. Synergistic, additive or antagonistic effects between
agents may be quantified by analysis of the dose-response curves
using the Combination Index (CI) method. A CI value greater than 1
indicates antagonism; a CI value equal to 1 indicates an additive
effect; and a CI value less than 1 indicates a synergistic effect.
In one embodiment, the CI value of a synergistic interaction is
less than 0.9. In another embodiment, the CI value is less than
0.8. In a preferred embodiment, the CI value is less than 0.7.
[0088] This invention provides a method of inhibiting HIV-2
infection of a susceptible cell by HIV-2 which comprises subjecting
the susceptible cell to an effective HIV-2 infection inhibiting
dose of (a) a humanized antibody designated PRO 140, or of (b) an
anti-CCR5 receptor monoclonal antibody which (i) binds to CD4+CCR5+
cells and inhibits fusion of HIV-1 with such cells, (ii) inhibits
HIV-1 fusion with CD4+CCR5+ cells with a potency equal or greater
than that of PRO 140, (iii) coats CD4+CCR5+ cells in the subject
without reducing the number of such cells in the subject, and/or
(iv) binds to the subject's CD4+CCR5+ cells without inducing an
increase in the subject's plasma concentration of circulating
.beta.-chemokines, wherein PRO 140 comprises (i) two light chains,
each light chain comprising the light chain variable (V.sub.L) and
constant (C.sub.L) regions encoded by the plasmid designated
pVK:HuPRO140-VK (ATCC Deposit Designation PTA-4097), and (ii) two
heavy chains, each heavy chain comprising the heavy chain variable
(V.sub.H) and constant (C.sub.H) regions encoded either by the
plasmid designated pVg4:HuPRO140 HG2-VH (ATCC Deposit Designation
PTA-4098) or by the plasmid designated pVg4:HuPRO140 (mut B+D+I)-VH
(ATCC Deposit Designation PTA-4099), wherein the effective HIV-2
infection inhibiting dose comprises from 0.1 mg per kg to 25 mg per
kg of the subject's body weight, so as to thereby inhibit the
infection of the susceptible cell by HIV-2. The susceptible cell
may be present in a human subject. The anti-CCR5 receptor
monoclonal antibody may bind to the same CCR5 epitope as that to
which PRO 140 binds. The anti-CCR5 receptor monoclonal antibody may
be a humanized, a human, or a chimeric antibody. The susceptible
cell may be subject to an effective HIV-2 infection inhibiting dose
of the antibody designated PRO 140. The antibody designated PRO 140
comprises (i) two light chains, each light chain comprising the
light chain variable (V.sub.L) and constant (C.sub.L) regions
encoded by the plasmid designated pVK:HuPRO140-VK (ATCC Deposit
Designation PTA-4097) and (ii) two heavy chains, each heavy chain
comprising the heavy chain variable (V.sub.H) and constant
(C.sub.H) regions encoded by the plasmid designated pVg4:HuPRO140
HG2-VH (ATCC Deposit Designation PTA-4098). In the instant methods,
HIV-2 may be of a subtype selected from subtypes A, B, C, D, E, F,
G, H, J, O, or a combination thereof.
[0089] This invention also provides a method of inhibiting HIV-2
infection in an HIV-2-infected human subject which comprises
administering to the subject an effective HIV-2 infection
inhibiting dose of (a) a humanized antibody designated PRO 140, or
of (b) an anti-CCR5 receptor monoclonal antibody which (i) binds to
CD4+CCR5+ cells in the subject and inhibits fusion of HIV-1 with
such cells, (ii) inhibits HIV-1 fusion with CD4+CCR5+ cells with a
potency equal or greater than that of PRO 140, (iii) coats
CD4+CCR5+ cells in the subject without reducing the number of such
cells in the subject, and/or (iv) binds to the subject's CD4+CCR5+
cells without inducing an increase in the subject's plasma
concentration of circulating .beta.-chemokines, wherein PRO 140
comprises (i) two light chains, each light chain comprising the
light chain variable (V.sub.L) and constant (C.sub.L) regions
encoded by the plasmid designated pVK:HuPRO140-VK (ATCC Deposit
Designation PTA4097), and (ii) two heavy chains, each heavy chain
comprising the heavy chain variable (V.sub.H) and constant
(C.sub.H) regions encoded either by the plasmid designated
pVg4:HuPRO140 HG2-VH (ATCC Deposit Designation PTA-4098) or by the
plasmid designated pVg4:HuPRO140 (mut B+D+I)-VH (ATCC Deposit
Designation PTA-4099), wherein the effective HIV-2 infection
inhibiting dose comprises from 0.1 mg per kg to 25 mg per kg of the
subject's body weight, so as to thereby inhibit HIV-2 infection in
the subject. The susceptible cell may be present in a human
subject. The anti-CCR5 receptor monoclonal antibody may bind to the
same CCR5 epitope as that to which PRO 140 binds. The anti-CCR5
receptor monoclonal antibody may be a humanized, a human, or a
chimeric antibody. The susceptible cell may be subject to an
effective HIV-2 infection inhibiting dose of the antibody
designated PRO 140. The antibody designated PRO 140 comprises (i)
two light chains, each light chain comprising the light chain
variable (V.sub.L) and constant (C.sub.L) regions encoded by the
plasmid designated pVK:HuPRO140-VK (ATCC Deposit Designation
PTA-4097) and (ii) two heavy chains, each heavy chain comprising
the heavy chain variable (V.sub.H) and constant (C.sub.H) regions
encoded by the plasmid designated pVg4:HuPRO140 HG2-VH (ATCC
Deposit Designation PTA-4098). In the instant methods, the
effective HIV-2 infection inhibiting dose may be from 0.1 mg per kg
to 50 mg per kg, 0.25 mg per kg to 20 mg per kg of the subject's
body weight, from 0.5 mg per kg to 10 mg per kg of the subject's
body weight, or from 1 mg per kg to 5 mg per kg of the subject's
body weight. The effective HIV-2 infection inhibiting dose may be 5
mg per kg of the subject's body weight, 10 mg/kg of the subject's
body weight, or 20 mg/kg of the subject's body weight.
[0090] The effective HIV-2 infection inhibiting dose may be
administered at regular intervals. The effective HIV-2 infection
inhibiting dose may be sufficient to achieve in the subject a serum
concentration of the antibody of at least 400 ng/ml. The effective
HIV-2 infection inhibiting dose may be sufficient to achieve and
maintain in the subject a serum concentration of the antibody of at
least 1 .mu.g/ml. The effective HIV-2 infection inhibiting dose may
be sufficient to achieve and maintain in the subject a serum
concentration of the antibody of about 3 to about 12 .mu.g/ml. The
effective HIV-2 infection inhibiting dose may be sufficient to
achieve and maintain in the subject a serum concentration of the
antibody of at least 5 .mu.g/ml. The effective HIV-2 infection
inhibiting dose may be sufficient to achieve and maintain in the
subject a serum concentration of the antibody of at least 10
.mu.g/ml or at least 15 ug/ml, or at least 20 ug/ml. The effective
HIV-2 infection inhibiting dose may be sufficient to achieve and
maintain in the subject a serum concentration of the antibody of at
least 25 .mu.g/ml, or at least 30 ug/ml, or at least 35 ug/ml. The
effective HIV-2 infection inhibiting dose may be sufficient to
achieve and maintain in the subject a serum concentration of the
antibody of at least 50 .mu.g/ml. The effective HIV-2 infection
inhibiting dose may be administered at one or more predefined
intervals. The predefined interval may be at least once weekly,
every two to four weeks, every two weeks, every three weeks, is
every four weeks, at least once monthly, every six weeks, or every
eight weeks. In the instant methods, the resistant HIV-2 virus may
be of a subtype selected from subtypes A, B, C, D, E, F, G, H, J,
O, or a combination thereof. The antibody may be administered via
intravenous infusion or via subcutaneous injection.
[0091] The instant methods of the invention may further comprise
administering to the subject at least one additional antiretroviral
agent effective against HIV-2. The antiretroviral agent may be a
CCR5 antagonist that does not compete with the humanized antibody
designated PRO 140 of (a), or the anti-CCR5 receptor monoclonal
antibody of (b). The CCR5 antagonist may be an antibody. The
antibody may be a monoclonal antibody, a humanized antibody, a
human antibody, or a chimeric antibody. The CCR5 antagonist may be
a non-antibody, small-molecule CCR5 antagonist. The subject may be
treatment-naive or treatment-experienced.
[0092] This invention also provides a method of inhibiting in a
human subject the onset or progression of an HIV-2-associated
disorder, the inhibition of which is effected by inhibiting fusion
of HIV-2 which comprises administering to the subject at a
predefined interval effective fusion-inhibitory doses of a
humanized antibody designated PRO 140, or of an anti-CCR5 receptor
antibody which (i) binds to CD4+CCR5+ cells in the subject and
inhibits fusion of HIV-1 with such cells, (ii) inhibits HIV-1
fusion with the subject's CD4+CCR5+ cells with a potency
characterized by an IC90 of 10 .mu.g/ml or less, (iii) coats the
subject's CD4+CCR5+ cells without reducing the number of such cells
in the subject, and/or (iv) binds to the subject's CD4+CCR5+ cells
without inducing an increase in the subject's plasma concentration
of circulating .beta.-chemokines, wherein PRO 140 comprises (i) two
light chains, each light chain comprising the light chain variable
(V.sub.L) and constant (C.sub.L) regions encoded by the plasmid
designated pVK:HuPRO140-VK (ATCC Deposit Designation PTA-4097), and
(ii) two heavy chains, each heavy chain comprising the heavy chain
variable (V.sub.H) and constant (C.sub.H) regions encoded either by
the plasmid designated pVg4:HuPRO140 HG2-VH (ATCC Deposit
Designation PITA-4098) or by the plasmid designated pVg4:HuPRO140
(mut B+D+I)-VH (ATCC Deposit Designation PTA-4099), wherein each
administration of the antibody delivers to the subject from 0.1 mg
per kg to 25 mg per kg of the subject's body weight, so as to
thereby inhibit the onset or progression of the HIV-2-associated
disorder in the subject.
[0093] This invention further provides a method of reducing the
likelihood of a human subject's contracting infection by HIV-2
which comprises administering to the subject at a predefined
interval effective fusion-inhibitory doses of a humanized antibody
designated PRO 140, or of an anti-CCR5 receptor antibody which (i)
binds to CD4+CCR5+ cells in the subject and inhibits fusion of
HIV-1 with such cells, (ii) inhibits HIV-1 fusion with the
subject's CD4+CCR5+ cells with a potency characterized by an IC90
of 10 .mu.g/ml or less, (iii) coats the subject's CD4+CCR5+ cells
without reducing the number of such cells in the subject, and/or
(iv) binds to the subject's CD4+CCR5+ cells without inducing an
increase in the subject's plasma concentration of circulating
.beta.-chemokines, wherein PRO 140 comprises (i) two light chains,
each light chain comprising the light chain variable (V.sub.L) and
constant (C.sub.L) regions encoded by the plasmid designated
pVK:HuPRO140-VK (ATCC Deposit Designation PTA-4097), and (ii) two
heavy chains, each heavy chain comprising the heavy chain variable
(V.sub.H) and constant (C.sub.H) regions encoded either by the
plasmid designated pVg4:HuPRO140 HG2-VH (ATCC Deposit Designation
PTA-4098) or by the plasmid designated pVg4:HuPRO140 (mut B+D+I)-VH
(ATCC Deposit Designation PTA-4099), wherein each administration of
the antibody delivers to the subject from 0.1 mg per kg to 25 mg
per kg of the subject's body weight, so as to thereby reduce the
likelihood of the subject's contracting an HIV-2 infection. The
subject may have been exposed to HIV-2, or may be at risk of being
exposed to HIV-2.
[0094] The subject may be treatment-naive, i.e., the subject has
not previously undergone treatment with any anti-HIV-2,
antiretroviral agents. The subject may also be
treatment-experienced, i.e., the subject has undergone, and/or is
undergoing, treatment with one or more anti-HIV-2, antiretroviral
agents. Diagnostic assessment of subjects undergoing treatment with
PRO 140, alone or in combination with other antiretrovirals,
including other CCR5 receptor antagonists, are encompassed by this
invention. In an embodiment, a subject to be treated with PRO 140
is tested prior to treatment to assess the subject's HIV tropism.
Tropism refers to the affinity of a virus for a specific
co-receptor on a target cell. The subject may be
treatment-experienced or treatment-naive. Viral tropism may be
assessed or screened by procedures known in the art, such as the
Trofile.TM. Assay (Monogram Biosciences, South San Francisco,
Calif.), which can provide an HIV profile for a subject, i.e., the
strain of virus (R5, X4, or D/M (dual/mixed (R5/X4)) that infects
the subject. A subject determined to be infected with HIV-2 may
then undergo treatment with PRO 140. An embodiment of the invention
is therefore directed to a method of treating an HIV-1-infected
subject with PRO 140 to reduce viral load in the subject, wherein
the subject is diagnostically determined to be infected with
CCR5-tropic HIV-2 prior to treatment, and then is treated with PRO
140 in accordance with any of the treatment and dosing methods
described herein. In an embodiment, a subject is screened for CCR5
viral tropism about one to six weeks before treatment with PRO 140.
In an embodiment, a subject is screened for CCR5 viral tropism
about three to six weeks before treatment with PRO 140. In an
embodiment, a subject is screened for CCR5 viral tropism about two
to five weeks before treatment with PRO 140. In an embodiment, a
subject is screened for CCR5 viral tropism about a month to a month
and a half before treatment with PRO 140.
[0095] In another embodiment, a subject is monitored and screened
at repeated intervals during the course of treatment with PRO 140
to determine HIV tropism according to procedures known and used by
those skilled in the art. In this way, a subject's drug regimen
(e.g., dosing and/or co-administration of other antiretrovirals
with PRO 140) can be adjusted or modified as necessary or required
according to the subject's virus tropism profile over time. In an
embodiment, the subject is determined to be infected with HIV-2
prior to treatment with PRO 140, with or without other
antiretrovirals. Illustratively, monitoring of viral tropism in a
subject who is being treated with PRO 140, alone or in combination
with other antiretrovirals, may be maintained for a period of six
months, one year, two years, three years, four years, five years or
longer, as necessary, after treatment is begun. In an embodiment,
monitoring a subject for a change in viral tropism may be
correlated with other parameters, such as CD4 cell count and viral
load. For example, a change in treatment may not be warranted if a
change in tropism in a subject undergoing treatment occurs in the
absence of any effects on viral load or CD4 cell count in that
subject. In addition, a relative increase in X4 virus versus an
absolute increase in X4 virus in a patient being treated can be
assessed to determine optimization or assessment of a subject's HIV
treatment regimen. A relative increase in X4 tropic virus may
reflect an increased chance of detection, and may not be as
significant if observed during monitoring as an absolute increase
in X4 tropic virus, since an absolute increase in X4 tropic virus
may reflect a potentially preferential expansion of the X4 virus
population in the subject. One skilled in the art will further
appreciate that if a subject's CCR5-tropic HIV infection is being
inhibited and viral load is being reduced by the use of a CCR5
receptor antagonist, an increase in the detection of X4 virus, if
present, might be expected, even in the absence of any absolute
increase in the amount of X4 virus, as a result, for example, of
depletion of CCR5-tropic HIV. (Report of an FDA/FCHR Joint Public
Meeting, May 31, 2006, Forum for Collaborative HIV Research, Apr.
24, 2007). Thus, virus tropism monitoring should be conducted with
such outcomes in mind.
[0096] In an embodiment, a subject undergoing treatment with PRO
140, alone or in combination with other antiretroviral drugs, is
tested for HIV drug resistance at predetermined intervals during
the course of treatment. A non-limiting example of a widely used
phenotypic HIV drug resistance test is the PhenoSense.TM. HIV
assay, which measures the sensitivity of a virus to antiretroviral
drugs. For example, it has been found that the in vitro
susceptibility data obtained in the PhenoSense.TM. HIV Entry Assay
is in good agreement with data obtained from testing the same
patient-derived viral envelopes in PBL. Thus, this assay, or
similar assays, may be used as a primary screen for testing patient
samples for resistance to an antiretroviral CCR5 entry inhibitor. A
clinician or practitioner is able to determine the level of
susceptibility that a person has to each antiretroviral drug in
order to design an individualized treatment regimen. In addition,
such resistance testing and assessment may be continued in a
subject receiving PRO 140 as a treatment regimen, alone or in
combination with other antiretroviral drugs, to provide follow-up
of the treated subjects at predetermined intervals.
[0097] In an embodiment, subjects who are undergoing treatment with
PRO 140, alone, or in combination with other antiretroviral drugs,
which may include other CCR5 receptor antagonists, are monitored
for the development of tumors, e.g., lymphomas and sarcomas, and
malignancies at repeated intervals. Without limitation, such
intervals may be established to be, for example, once a month,
twice a month, once every three weeks, once every six weeks, once
every two to six months, or two to six times a year. In an
embodiment, subjects who are undergoing treatment with PRO 140,
alone or in combination with other antiretroviral drugs which may
include other CCR5 receptor antagonists, are monitored for the
development of infections (bacterial, viral, opportunistic, etc.).
Monitoring of subjects receiving treatment with one or more CCR5
receptor antagonists may include assessment, at the same or at
different times, of, for example, virus tropism changes, viral
resistance, viral load (HIV RNA levels), CD4 cell count and
tumor/malignancies, etc., at repeated intervals during the
treatment, e.g., on a monthly basis, every six weeks, every eight
weeks, every ten weeks, every twelve weeks, or 2-3 times per year.
Such assessments further involve the storage of baseline samples,
e.g., serum, taken from the subject prior to and/or at the time of
beginning a treatment regimen. Additionally, molecular clonal
analysis of the virus population(s) in a subject at baseline may be
assessed using methods known and practiced in the art. In this way
it can be determined that any tropism change in a subject's virus
population (e.g., a CXCR4 variant or dual/mixed virus) emerged from
a pre-existing reservoir in the subject not detected at baseline
and not from a co-receptor use change in the subject. For each of
the above embodiments directed to follow-up, monitoring and
periodic screening of subjects undergoing treatment for HIV
infection, those skilled in the art will be able to determine the
appropriate time intervals in which such follow-up, monitoring and
screening assessments should be made.
[0098] In accordance with the various methods of the present
invention, it will be appreciated that the humanized anti-CCR5
monoclonal antibody PRO 140 possesses a distinct pattern of viral
resistance, synergizes with small molecule drugs, blocks HIV
without CCR5 antagonism in vitro, exhibits a potential for improved
tolerability, enables infrequent dosing and is not expected to be
involved in drug-drug or food interactions and is well tolerated in
human subjects based on preclinical studies as described
hereinbelow. Thus, PRO 140 is advantageously used alone or in
combination with other antiretroviral drugs or agents in methods of
treating HIV infection and in methods of reducing viral load in an
HIV infected patient.
Short Term, Interim, or Induction Use:
[0099] Either upon initiation of first HIV therapy regimen, or upon
switch of therapy (first line to second line, etc.) the objective
of antiviral therapy is to maximally suppress viral load as quickly
as possible. Use of PRO-140 in combination with other
antiretroviral drugs, even for a short period of time (+/-3
months), can help to ensure rapid and full viral suppression to
<50 copies (HIV RNA/ml3). Use of PRO-140 could be continued for
a minimum of 12 weeks or until full viral suppression (<50
copies) is achieved. Whether dosed once monthly IV or once weekly
subcutaneously, PRO-140 used in an induction format can assist in
rapidly suppressing viral replication, protecting the
susceptibility of concurrent HIV drugs, as well as sensitivity of
patient virus to subsequent HIV drugs. Use of PRO-140 in this
manner coincides with the current standard of care at the start of
HIV therapy, or upon treatment switching, where frequent viral load
testing is conducted (up to 1.times./week in the first month, and
or 1.times./month in the first three months), facilitating PRO-140
administration (e.g., monthly) at the time of clinic visits for
laboratory testing blood draws. The concept of
induction/maintenance is much like the model often used in cancer
of ablation upfront, followed by maintenance (lower/less intensive)
chemotherapy for a period thereafter. PRO-140 is used for a short
period of time, say 3-6 months, in combination with other
anti-retrovirals (or alone) in order to rapidly and completely
suppress HIV viral replication and stimulate CD4+ cell
proliferation. Once desired levels are achieved and confirmed
through repeated lab tests (2 viral load tests indicating <50
copies/ml3 and/or >100 CD4+ cell increase), PRO-140 use could be
stopped, while patients continue with other anti-retroviral agents
to maintain these levels of suppression and CD4+ immune system
status.
[0100] PRO-140 "intensifies" the potency/effectiveness of an
antiretroviral regimen, for patients who are either new (naive) to
therapy or those who are switching therapy due to inadequate
virologic or immunologic response to prior therapy. PRO-140 would
be used in an acute and temporary manner with this approach to
achieve a desired result and then cease using it, rather than using
it chronically even after an endpoint is met as with most anti-HIV
drugs today. Being able to dose PRO-140 once every month helps to
render this approach more feasible as it coincides with normal
blood draws following HIV therapy initiation or switch.
[0101] PRO 140 can be administered to HIV-2 infected patients who
are transitioning from one drug regimen to another. PRO 140 can be
administered to the patient during the interim time period between
one drug regimen and a second drug regimen of different drugs, or
different drug combinations, and/or different drug doses, etc.
PRO-140 can safely be removed from the combination of anti-HIV
drugs used to achieve full suppression, once viral load has reached
<50 copies following two separate lab tests. This is the case
where at least two, but preferably three, active drugs are used in
the follow-on (maintenance or subsequent) regimen.
Intermittent Viral Load Detection
[0102] Temporary use of PRO-140 is also appropriate in cases where
patients have viral load that is generally suppressed to <50 or
<400 copies, but occasionally rises to levels exceeding these
thresholds. Use of PRO-140 for one to three months following two
viral load tests confirming `viral escape` may support the patients
current HIV therapy and effectively re-suppress viral replication.
Use of PRO-140 even in this short term modality may also afford
important immune system restoration function in the form of CD4+
proliferation to further improve patients clinical status.
Persistent, Low level Viral Replication
[0103] It is common among treatment experienced patients to see
incomplete viral suppression, or stable, low-level viral
replication (>400 but <10,000 copies). In such cases,
clinicians often allow patients to continue their HIV regimen as
long as there is no change in clinical status or CD4+ count.
Temporary (+/-3 month) use of PRO-140 may assist clinicians in
suppressing viral loads to <50 copies, even in patients who have
never reached this objective, with or without changing some/all of
the patients other concurrent anti-HIV medications. Use of PRO-140
even in this short term modality may also afford important immune
system restoration function in the form of CD4+ proliferation to
further improve patients clinical status.
Boosting CD4 Count in Patients with CCR5, Dual Mixed or CXCR4
Tropic Virus
[0104] In treatment experienced patients or patients infected with
multi-drug resistant HIV virus, often the primary goal of therapy
is not to suppress HIV viral load but to sustain or improve immune
system (CD4+ cell) function. Use of PRO-140 in such patients,
either alone or in combination with other antiretroviral agents and
regardless of HIV virus tropism, may help boost CD4+ cell count and
stabilize a patient's clinical status thus reducing the risk of HIV
disease progression.
Re-Use or Recycling of PRO-140
[0105] Other entry inhibitors, specifically enfuvirtide, have
published data demonstrating that the drug may have residual
activity in up to 50% of patients who have documented resistance
and treatment failure to that drug. Following discontinuation of
enfuvirtide therapy for 60-90 days, genetic mutations in the gp41
envelope region (36-45) appear to revert to wild-type status. Upon
re-initiation of enfuvirtide therapy, up to 50% of patients achieve
a response in viral load reduction of .about.1 log which is
sustained for at least 6 months. Among patients who reinitiated
enfuvirtide therapy and did not respond, resistance mutations in
the gp41 envelope region differed from those seen in prior
enfuvirtide therapy. PRO-140 possesses the same characteristics in
inducing conformational changes in the V3 loop region, that differ
upon reintroduction of drug following prior documented resistance
and treatment failure. This renders recycling or reuse of PRO-140 a
viable therapeutic approach.
Extra-Cellular Only HIV Regimens
[0106] With the development of numerous anti-HIV compounds whose
mechanism of action focuses on prevention of HIV virus entry into
the target (CD4+) immune system cells, it may be possible to fully
suppress HIV replication by using combinations of such
extra-cellularly active drugs alone. This implies the potential for
simplified HIV regimens (fewer drugs needed) that block viral entry
and "protect" immune system cells. This approach also has the
potential to reduce drug interactions, drug related toxicities as
well as exacerbation of co-morbidities often seen in HIV patients
(hepatitis, etc.). Use of PRO-140 with other extracellularly active
anti-HIV drugs that target either HIV or host proteins (including
gp41 fusion inhibitors, CCR5, CXCR4, gp120 or other moieties) could
be sufficient to fully suppress HIV replication in a sustained
manner. This would avoid the need for co-administration of NRTIs,
nNRTI's, protease inhibitors (PIs) or integrase inhibitors with
PRO-140.
Leveraging Synergistic Mechanistic Activity with PIs
[0107] Evidence exists to support the selective use of HIV viral
entry inhibitors with ritonavir boosted protease inhibitors to
achieve synergistic MOA based activity that results in enhanced
viral suppression compared to combinations of anti-HIV drugs from
other classes. By preventing HIV viral entry in to CD4+ cells, as
well as preventing HIV viral expression from CD4+ cells after
intracellular incorporation, PRO-140 and protease inhibitors may
induce greater, and/or more rapid, and/or more complete HIV viral
suppression than the combination of other mechanisms of action.
This synergy could provide the rationale to preferentially use
PRO-140 with protease inhibitors, with or without other anti-HIV
drugs, to achieve maximal viral suppression and CD4+
proliferation.
Co-Formulation with Other Anti-HIV Drugs
[0108] Based on the above information, as well as the established
precedent in HIV therapy to date, ample rational exists for the
co-formulation of PRO-140 with other anti-HIV drugs to enable
combined administration. Such co-formulation could involve other
injectable anti-HIV drugs or oral anti-HIV drugs that are
reformulated into parenteral forms.
PRO-1140 Use to Impair Viral Fitness and Pathogenicity
[0109] Use of PRO-140 in either lower (than therapeutically
necessary) doses or less frequently (dosed) in order to exert
sufficient pressure that forces the HIV virus to mutate and reduces
the efficacy of PRO-140; however, as a result, a virus that is less
virulent, pathogenic or `fit` (less capable to replicate) is
produced. This might be an application suited for patients whose
virus has developed resistance to PRO-140, but who are still
deriving some type of immunologic benefit (sustained or rising
levels of CD4+ cells--also termed discordant response) and thus may
still derive benefit from continuing PRO-140 therapeutic treatment.
Such a debilitated HIV virus may also be more susceptible to other
HIV drugs, improving their effect on HIV viral suppression or CD4+
response.
PRO-140 Use in Immune Cell Mobilization
[0110] Given the early and robust proliferation in CD4+ cells from
the Phase 1b study, it is possible that PRO-140 exhibits a
mechanism of action that potently effect both active and resting
CD4+ cells, as well as other immune system cells, in a manner that
is different from other entry inhibitors. Use of PRO-140 in single
or multiple doses to stimulate or accelerate immune system cell
proliferation may be appropriate and justified, whether in HIV
infected patients, whether naive to HIV therapy, currently on
therapy or who have ceased HIV therapy due to resistance or other
reasons.
[0111] The following Experimental Details are set forth to aid in
an understanding of the subject matter of this disclosure, but are
not intended to, and should not be construed to, limit in any way
the claims which follow thereafter.
EXPERIMENTAL DETAILS
Part I
Materials and Methods
[0112] Compounds and mAbs
[0113] PRO 140 was prepared by expression in Sp2/0 cells using
Hybridoma serum-free medium supplemented with 2 mM L-glutamine
(Invitrogen, Carlsbad, Calif.). Bulk mAb was clarified using a 5.0
.mu.m Depth filter (Sartorius, Goettingen, Germany) followed by
passage over a 0.2 .mu.m sterilizing grade filter (Sartorius). The
mAb was purified by passage first over an affinity column
(MabSelect Protein A column, Amersham, Piscataway, N.J.) and then
by ion exchange chromatography (SP Sepharose Cation Exchange resin,
Amersham). PRO 140 was nanofiltered using a Viresolve.TM. 10
Opticap NFP capsule (Millipore, Billerica, Mass.) followed by a 0.2
.mu.m filter and concentrated/diafiltered over disposable TFF
cartridges (Millipore). The mAb was then polished over a
hydroxyapatite column (Bio-Rad, Hercules, Calif.), concentrated to
10 mg/ml in phosphate-buffered saline and stored at -70.degree. C.
or colder prior to use.
[0114] RANTES was purchased from R&D Systems (Minneapolis,
Minn.). The anti-CCR5 mAb 2D7 was purchased from BD Biosciences
(Cat. #555993), and the anti-CCR5 mAb CTC5 was purchased from
R&D Systems (Cat. #FAB1802P).
RET Assay
[0115] The HIV-1 RET assay has been described in detail previously
(Litwin et al., 1996). Briefly, fluorescein octadecyl ester (F18;
Molecular Probes, Eugene, Oreg.; 5 mg/ml in ethanol), was diluted
1:800 in DMEM labeling medium (DMEM; Invitrogen, Carlsbad, Calif.)
with 10% fetal bovine serum (FBS; HyClone, Logan, Utah) and
adjusted to an A.sub.506 of 0.34.+-.10%. Octadecyl rhodamine B
chloride (R18; Molecular Probes; 10 mg/ml in ethanol) was diluted
1:2050 in labeling medium and adjusted to an A.sub.565 of
0.52.+-.10%. Both dyes were further diluted 2-fold by addition to
cells in T75-cm.sup.2 flasks. HeLa-Env.sub.JRFL and CEM NKR-CCR5
cells were incubated overnight in F18- and R18-containing culture
medium, respectively. The following day, medium from
HeLa-Env.sub.JRFL cells was removed and 10 ml of 0.5 mM EDTA was
added and incubated at 37.degree. C. for 5 min. EDTA was removed
and the flask was returned to the incubator for another 5 min
followed by striking of the flask to dislodge cells. Ten ml of PBS-
with 15% FBS were added to the flask and the contents were
transferred to a 50-ml conical centrifuge tube. Suspension CEM
NKR-CCR5 cells were added directly to a separate 50-ml conical
centrifuge tube. Both cell lines were centrifuged at 300.times.g
for 5 min. The supernatant was discarded and cells were resuspended
in 10 ml of PBS-/15% FBS. The centrifugation/wash step was repeated
twice, after which the cells were counted and concentrations
adjusted to 1.5.times.10.sup.6 cells/ml. Ten .mu.l of each cell
type (15,000 cells) were seeded into wells of a 384-well plate.
Inhibitor compounds were added immediately thereafter to bring the
final well volume to 40 .mu.l, and the plates were incubated for 4
h at 37.degree. C. Compounds were tested individually and in
combination at a fixed molar ratio or mass ratio over a range of
serial dilutions. The plates were then read on a fluorescence plate
reader (Victor.sup.2, Perkin Elmer, Boston, Mass.) using the
excitation/emission filter combinations shown in Table 6.
TABLE-US-00003 TABLE 6 Excitation/emission filter combinations for
RET assay Scan No. Excitation wavelength Emission wavelength 1 450
nm/50 nm 530 nm/25 nm 2 530 nm/25 nm 590 nm/35 nm 3 450 nm/50 nm
590 nm/35 nm
[0116] The "% RET" was calculated according to the following
formula after subtraction of background (blank) readings:
%
RET=100.times.[(A.sub.3-(A.sub.1.times.F.sub.spill)-(A.sub.2.times.R.s-
ub.spill))/A.sub.2]
Where:
[0117] F.sub.spill=HeLa cells alone, Scan 3/Scan 1; [0118]
R.sub.spill=CEM cells alone, Scan 3/Scan 2; [0119] A.sub.1=Scan 1
value for HeLa and CEM cells in combination; [0120] A.sub.2=Scan 2
value for HeLa and CEM cells in combination; and [0121]
A.sub.3=Scan 3 value for HeLa and CEM cells in combination.
[0122] The "% Inhibition" was calculated according to the following
formula:
% Inhibition=100.times.[(Max % RET-% RET for sample well)/(Max %
RET-Min % RET)]
Where:
[0123] Max % RET=average of % RET values for HeLa and CEM cell
combination without added inhibitor; and [0124] Min % RET=average
of % RET values for HeLa and CEM cell combination in presence of
500 ng/ml of Leu-3a mAb (an antibody that targets CD4 and fully
blocks fusion in the RET assay at this concentration).
[0125] Fifty percent inhibition (IC.sub.50) values were determined
by fitting the inhibition data with a non-linear, four-parameter,
variable slope equation (GraphPad Prism, 4.02; GraphPad Software,
San Diego, Calif.). Upper and lower inhibition values were
constrained to 100% and 0%, respectively for curve fitting.
Preparation of PBMCs
[0126] Replication of authentic HIV-1 is measured in activated
peripheral blood mononuclear cells (PBMCs) using the
monocyte/macrophage-tropic HIV-1 clone, JRFL (HIV-1.sub.JRFL), for
these studies.
[0127] PBMCs are isolated from 4 separate donors (Leukopacks) by
centrifugation on a Ficoll gradient. CD8 cells are depleted using
RosetteSep CD8 Depletion Cocktail (#15663, StemCell Research,
Vancouver, BC). Cells are diluted to 4.times.10.sup.6/ml and added
in equal parts to three T175-cm.sup.2 flasks and then stimulated by
addition of one of the following media: IL-2 Medium [RPMI 1640
(#10-040-CV, Cellgro, Herndon, Va.), 10% FBS (#35-010-CV), 2 mM
L-Glutamine (#25-005-CI), 100 U/ml IL-2 (Sigma, St. Louis, Mo.)];
PHA 5 Medium: [IL-2 Medium with 5 ug/ml Phytohemagglutinin PHA-P
(PHA) (#L8754, Sigma, St. Louis, Mo.), filtered]; or PHA 0.5
Medium: [IL-2 Medium with 0.5 ug/ml PHA, filtered]. Each flask
receives a total of 50-150 ml of medium. Flasks are incubated for 3
days at 37.degree. C. followed by pooling of the contents prior to
use in the infection assay.
Virus Titration
[0128] Serial dilutions of virus are tested in quadruplicate on
activated PBMCs (1.4.times.10.sup.5 PBMC/well). Titration Medium
[IL-2 Medium with 100 IU/ml penicillin/streptomycin (#30-002-CI,
Cellgro)] is utilized for virus titrations. Fifty .mu.l of diluted
virus is added to 100 .mu.l of PBMCs in flat bottom, tissue-culture
treated 96-well plates (VWR# 29442-054, Corning, Corning, N.Y.) and
the plates are incubated at 37.degree. C. in a humidified, 5%
CO.sub.2 incubator. After 7 days, 50 .mu.l are removed from each
well and tested for virus levels by p24 antigen ELISA (Perkin
Elmer, Boston, Mass.). Virus titer is determined by the method of
Reed and Muench (Table 11, see below).
Neutralization Assay
[0129] Stimulated PBMCs are seeded into wells of 96-well flat
bottom plates at a density of 1.4.times.10.sup.5 cells/well. Virus
is diluted to 2,000 TCID.sub.50/ml and mixed with serial 0.5
log.sub.10 dilutions of compound for 1 h at 37.degree. C. prior to
addition to the cell plates. The final amount of virus added per
well is 100 TCID.sub.50. The final DMSO concentration in the assay
is always 0.5% whenever small molecule inhibitors are being tested.
Plates are incubated at 37.degree. C. for 5 days, at which time an
aliquot of supernatant is removed for p24 antigen ELISA. If control
wells (virus without inhibitor) exhibit low p24 antigen levels then
the plates are brought back to full volume with Titration medium
and incubated for an additional 24 h.
Data Analysis
[0130] Neutralization activity is displayed by plotting the percent
inhibition of p24 antigen production (after background values are
subtracted from all datapoints) versus log.sub.10 drug
concentration. The percent inhibition is derived as follows [1-(p24
levels in the presence of drug/p24 levels in the absence of
drug)].times.100. IC.sub.50 values are determined by fitting the
inhibition data with a non-linear, four-parameter, variable slope
equation (GraphPad Prism, ver. 4.02; GraphPad Software, San Diego,
Calif.). Upper and lower inhibition values are constrained to 100%
and 0%, respectively for curve fitting.
Phase 1a Clinical Study
[0131] Individuals were treated in sequential, dose-rising cohorts
of 5 subjects (4 active and 1 placebo) each and evaluated for up to
120 days post-treatment. A population of healthy, i.e., HIV-1
uninfected, male volunteers with no abnormal findings on physical
exam, medical history and ECG, aged 19-50, was administered a
single intravenous infusion of PRO 140 (0.1, 0.5, 2.0 and 5.0 mg
per kg body weight). Safety assessments consisted of monitoring the
following: vital signs (blood pressure, pulse, temperature, etc;
hematology (hemoglobin, hematocrit, leukocytes, platelets, etc.);
serum chemistries (AST/ALT, alkaline phosphatase, BUN, creatinine,
etc.); urinalysis (pH, specific gravity, protein, glucose,
leukocytes, etc.); and ECGs (12-lead).
Measurement of Coating of CCR5 Cells by PRO 140
[0132] Whole blood specimens were combined separately with the
indicated phycoerythrin-labeled anti-CCR5 antibodies or with
appropriate isotype-control antibodies. Erythocytes were lysed and
leukocytes were stabilized using the ImmunoPrep Reagent System
(Beckman Coulter), and the cells were analyzed on a TQ Prep.TM.
flow cytometry workstation (Beckman Coulter). Data were expressed
as the percent of CCR5 cells relative to all cells gated in the
analysis. CTC5 is an anti-CCR5 antibody that does not compete with
PRO 140. 2D7 is an anti-CCR5 antibody that does compete with PRO
140.
Measurement of Serum Concentrations of PRO 140
[0133] Sera were diluted as appropriate and combined with L1.2-CCR5
cells, which are mouse pre-B lymphoma cells engineered to stably
express human CCR5. In order to generate a standard curve, PRO 140
standard was tested in parallel at concentrations ranging from
0.062 to 4.0 .mu.g/ml in 10% normal human serum (NHS). 10% NHS
containing no PRO 140 was analyzed as a negative control. Following
incubation with test samples, cells were washed and combined with a
FITC-labeled sheep antibody against human IgG4 (The Binding Site
Limited, Cat. #AF009). Cells were washed again and analyzed by flow
cytometry. The concentration of PRO 140 was determined by comparing
the median fluorescence intensity (MFI) of the test sample with MFI
values of the standard curve.
Determination of Plasma RANTES Concentration
[0134] The assay employed the Quantikine.TM. Human RANTES
Immunoassay Kit (R&D Systems, Minneapolis, Minn.). Briefly,
platelet-poor plasma was collected in CTAD/EDTA tubes and stored at
-20.degree. C. Test samples and RANTES standard were added to
microtiter plates that were pre-coated with a mouse monoclonal
antibody to RANTES. Following incubation, plates were washed and
contacted with an anti-RANTES polyclonal antibody conjugated to
horseradish peroxidase (HRP). Plates were washed again prior to
addition of tetramethlybenzidine substrate for colorimetric
detection. The Lower Limit of Quantification of the assay was 415
pg RANTES/ml plasma.
Results and Discussion
[0135] PRO 140 is a humanized IgG4,K anti-CCR5 mAb being developed
for HIV-1 therapy. This antibody has been shown to broadly and
potently inhibit CCR5-mediated fusion of HIV-1 to target cells in
vitro. PRO 140 is also highly active in a therapeutic hu-PBL-SCID
mouse model, and preliminary data are now available from a Phase 1a
clinical study in healthy human subjects.
In Vitro Antiviral Activity of PRO 140
[0136] Murine and humanized PRO 140 were tested against four
primary R5 HIV-1 isolates as described in the Methods. FIG. 1 shows
that PRO 140 has potent antiviral activity in vitro, neutralizing a
variety of primary R5 strains with an IC90 of 3-4 .mu.g/ml. PRO 140
exhibited similar antiviral activity to the murine mAb, PA14, from
which PRO 140 is derived.
Preliminary Data from Phase 1a Clinical Study
[0137] The primary objective of the Phase 1a study was to evaluate
the safety and tolerability of PRO 140 given as a single dose in a
rising dose cohort regimen in healthy male subjects. The secondary
objectives were (1) to gain information about the pharmacokinetics
of intravenously administered PRO 140, and (2) to gain information
on the effects of PRO 140 on blood levels of CCR5+ cells and
chemokines.
Pharmacokinetics of PRO 140
[0138] Healthy male volunteers were treated with a single
intravenous infusion of PRO 140 at dose levels of 0.1, 0.5, 2.0 and
5.0 mg/kg. PRO 140 and placebo were generally well tolerated with
no significant changes in ECGs and no dose-limiting toxicity.
[0139] Serum was collected post-treatment, cryopreserved, and
analyzed for PRO 140 levels. Peak serum concentrations ranged to 3
mg/ml at 0.1 mg/kg and 12 mg/ml at 0.5 mg/kg. Serum concentrations
remained detectable (>400 ng/ml for up to 5 days at 0.1 mg/kg,
21 days at 0.5 mg/kg, and for over 60 days following a single 2
mg/kg injection (FIG. 7). Serum concentrations of PRO 140 increased
proportionally with dose level, and the clearance rate was similar
to that of other humanized mAbs. Pharmacokinetic (PK) metrics were
determined using WinNonLin (PharSight Corporation, Mountain View,
Calif.) using a noncompartmental model, and the terminal serum
half-life of PRO 140 was determined to be 10-12 days. As expected,
no subject developed antibodies to the humanized PRO 140.
Coating and Non-Depletion of CCR5 Lymphocytes by PRO 140
[0140] Healthy male volunteers (n=4) were treated with a single
intravenous infusion of PRO 140 at a dose level of 2 mg/kg. For up
to 60 days post-treatment, at the times indicated in FIG. 6, blood
was collected and analyzed for CCR5 lymphocyte levels.
[0141] Following treatment with PRO 140, there was no decrease in
the overall number of CCR5 lymphocytes at measured by CTC5 binding;
however, the binding of antibody 2D7 was significantly decreased
(FIG. 6). Background binding of isotype control antibodies was
unchanged. Since the binding of CTC5 is not decreased by the
presence of PRO 140, the CTC5-PE values are a measure of the total
number of circulating CCR5 lymphocytes. Since 2D7 competes with PRO
140, the 2D7-PE values reflect the number of CCR5 lymphocytes that
are not coated with PRO 140.
[0142] The data indicate that a single 2 mg/kg dose of PRO 140
effectively coats CCR5 lymphocytes without cellular depletion for
two weeks, and cells remain partially coated for >4 weeks. In
addition, CCR5 coating was more prolonged in patients treated with
5 mg/kg PRO 140. The data indicate that a single 5 mg/kg dose of
PRO 140 effectively coats CCR5 lymphocytes without cellular
depletion and the cells remain partially coated for >60 days
(FIG. 10). Since CCR5 coating is the mechanism whereby PRO 140
inhibits HIV, viral loads in HIV-infected individuals could be
expected to decrease in a similar temporal manner.
Effect of PRO 140 on Plasma Chemokine Levels
[0143] Healthy male volunteers were treated with a single
intravenous infusion of 0.1 mg/kg PRO 140 (Cohort 1), 0.5 mg/kg PRO
140 (Cohort 2) or matched placebo. Plasma was collected
post-treatment at the indicated times, cryopreserved and analyzed
for levels of RANTES, a CC-chemokine that serves as a natural
ligand for CCR5. RANTES levels were measured by ELISA in
platelet-depleted plasma pre-dose and up to 28 days post-dose. As
shown in FIG. 8, there was no significant change in RANTES levels
following PRO 140 treatment (P>0.14 all times). These data are
consistent with in vitro findings that PRO 140 does not antagonize
CCR5 function. The findings suggest that PRO 140 does not have
untoward effects on CCR5-mediated immune function in treated
patients.
[0144] The results described herein indicate that in addition to
PRO 140 broadly and potently inhibiting CCR5-mediated HIV-1 entry
without CCR5 antagonism or other immunologic side effects in
preclinical testing, this has demonstrated favorable tolerability,
PK and immunologic profiles in preliminary results from an ongoing
Phase 1a study in healthy volunteers. Thus, in many respects, PRO
140 offers a novel and attractive product profile for anti-HIV-1
therapy.
[0145] Moreover, the activities of anti-CCR5 mAbs are fundamentally
distinct from, but complementary to, those of small-molecule CCR5
antagonists (see Table 2) which are also currently undergoing human
clinical trials. PRO 140 has recently been shown to work
synergistically with non-antibody CCR5 antagonists in inhibiting
CCR5-mediated HIV-1 fusion to target cells. Accordingly,
combination therapy comprising administration of anti-CCR5 mAbs and
non-antibody CCR5 antagonists may offer powerfully effective, new
approaches to preventing and treating HIV-1 infection.
Part II
Example 1
Combination Testing of PRO 140 and HIV-1 Entry Inhibitors in the
Fluorescence RET Assay
Materials and Methods
[0146] Compounds and mAbs
[0147] PRO 140 was prepared by expression in Sp2/0 cells using
Hybridoma serum-free medium supplemented with 2 mM L-glutamine
(Invitrogen, Carlsbad, Calif.). Bulk mAb was clarified using a 5.0
.mu.m Depth filter (Sartorius, Goettingen, Germany) followed by
passage over a 0.2 .mu.m sterilizing grade filter (Sartorius). The
mAb was purified by passage first over an affinity column
(MabSelect Protein A column, Amersham, Piscataway, N.J.) and then
by ion exchange chromatography (SP Sepharose Cation Exchange resin,
Amersham). PRO 140 was nanofiltered using a Viresolve.TM. 10
Opticap NFP capsule (Millipore, Billerica, Mass.) followed by a 0.2
.mu.m filter and concentrated/diafiltered over disposable TFF
cartridges (Millipore). The mAb was then polished over a
hydroxyapatite column (Bio-Rad, Hercules, Calif.), concentrated to
10 mg/ml in phosphate-buffered saline and stored at -70.degree. C.
or colder prior to use. SCH-D (Schering Plough; Tagat et al.,
2004), TAK-779 (Takeda Pharmaceuticals; Shiraishi et al., 2000),
UK427,857 (Pfizer; Wood and Armour, 2005), and BMS378806
(Bristol-Myers Squibb; Lin et al., 2003) were prepared by
commercial sources.
[0148] SCH-D has the following structure:
##STR00001## [0149] SCH-D (also designated SCH-417690):
1-[(4,6-dimethyl-5-pyrimidinyl)carbonyl]-4-[4-[2-methoxy-1(R)-4-(trifluor-
omethyl)phenyl]ethyl-3(S)-methyl-1-piperazinyl]-4-methylpiperidine
(Schering-Plough)
[0150] SCH-D was synthesized according to the procedure described
in Tagat et al. (2004) and set forth in FIG. 1.
[0151] TAK-779 has the following structure:
Y.dbd.--CH.sub.2
X.dbd.--Cl
R.sup.1.dbd.--CH.sub.3
##STR00002##
[0152] TAK-779 was synthesized according to the procedure described
in Shiraishi et al. (2000) and set forth in FIG. 2.
[0153] TAK-652 has the following structure:
##STR00003##
[0154] UK427,857 (maraviroc) has the following structure:
##STR00004##
[0155] UK427,857 was synthesized according to the procedure
described in PCT International Publication No. WO 01/90106 and set
forth in FIG. 3.
[0156] BMS378806 has the following structure:
##STR00005## [0157] BMS378806:
(R)-N-(benzoyl)-3-methyl-N'-[(4-methoxy-7-azaindol-3-yl)-oxoacetyl]-piper-
azine (Bristol-Myers Squibb)
[0158] It was synthesized according to the procedure described in
U.S. Pat. No. 6,476,034 (compound 17a). Nevirapine (Boehringer
Ingelheim; Merluzzi et al., 1990) and atazanavir (Bristol-Myers
Squibb; Robinson et al., 2000) were purchased from commercial
sources. PRO 542 was expressed in Chinese hamster ovary cells and
purified as described previously (Allaway et al., 1995). T-20
(Fuzeon.RTM.) was synthesized by solid-phase
fluoroenylmethoxycarbonyl chemistry, was purified by reverse-phase
chromatography and was analyzed for purity and size by HPLC and
mass spectroscopy as described previously (Nagashima et al., 2001).
AZT was purchased from Sigma Chemicals (St. Louis, Mo.). RANTES was
purchased from R&D Systems (Minneapolis, Minn.). The anti-CCR5
mAb 2D7 was purchased from Pharmingen (San Diego, Calif.), and the
anti-CD4 mAb Leu-3A was purchased from Becton Dickinson (Franklin
Lakes, N.J.).
[0159] For testing, small molecule compounds were solubilized in
dimethylsulfoxide (DMSO) to 10 mM and then diluted in DMSO to
200.times. the final concentration to be utilized in the antiviral
assay. Serial dilutions of small molecules were conducted in DMSO.
Subsequent dilutions were conducted in medium to achieve a final
DMSO concentration in the assay of 0.5%. Peptides and mAbs were
diluted in PBS in the absence of DMSO. Typically, inhibitor
concentrations in the RET assay included eleven 3-fold dilutions
ranging from 200 nM to 3.0 .mu.M.
Cell Preparation
[0160] HeLa cells were engineered to express HIV-1 gp120/gp41 from
the macrophage-tropic primary isolate JRFL as described
(HeLa-Env.sub.JRFL; Litwin et al., 1996). Briefly, the
HIV-1.sub.LAI Env gene was excised from the plasmid pMA243 (Dragic
et al., 1992) and the HIV-1.sub.JRFL Env gene was inserted. The
HIV-1.sub.JRFL Env gene was amplified from the plasmid pUCFL112-1
(Koyanagi et al., 1987). The resulting plasmid, designated
JR-FL-pMA243, was sequenced by standard methods and transfected
into HeLa cells using lipofectin (Gibco BRL/Invitrogen, Carlsbad,
Calif.). HeLa-Env.sub.JRFL transfectants were selected in
methotrexate (Sigma, St. Louis, Mo.) and cloned twice by limiting
dilution. The transduced human T cell leukemia line CEM NKR-CCR5
cells were obtained from the NIH AIDS Research and Reference
Program (Cat. No. 458).
RET Assay
[0161] The HIV-1 RET assay has been described in detail previously
(Litwin et al., 1996). Briefly, fluorescein octadecyl ester (F18;
Molecular Probes, Eugene, Oreg.; 5 mg/ml in ethanol), was diluted
1:800 in DMEM labeling medium (DMEM; Invitrogen, Carlsbad, Calif.)
with 10% fetal bovine serum (FBS; HyClone, Logan, Utah) and
adjusted to an A.sub.506 of 0.34.+-.10%. Octadecyl rhodamine B
chloride (R18; Molecular Probes; 10 mg/ml in ethanol) was diluted
1:2050 in labeling medium and adjusted to an A.sub.565 of
0.52.+-.10%. Both dyes were further diluted 2-fold by addition to
cells in T75-cm.sup.2 flasks. HeLa-Env.sub.JRFL and CEM NKR-CCR5
cells were incubated overnight in F18- and R18-containing culture
medium, respectively. The following day, medium from
HeLa-Env.sub.JRFL cells was removed and 10 ml of 0.5 mM EDTA was
added and incubated at 37.degree. C. for 5 min. EDTA was removed
and the flask was returned to the incubator for another 5 min
followed by striking of the flask to dislodge cells. Ten ml of PBS-
with 15% FBS were added to the flask and the contents were
transferred to a 50-ml conical centrifuge tube. Suspension CEM
NKR-CCR5 cells were added directly to a separate 50-ml conical
centrifuge tube. Both cell lines were centrifuged at 300.times.g
for 5 min. The supernatant was discarded and cells were resuspended
in 10 ml of PBS-/15% FBS. The centrifugation/wash step was repeated
twice, after which the cells were counted and concentrations
adjusted to 1.5.times.10.sup.6 cells/ml. Ten .mu.l of each cell
type (15,000 cells) were seeded into wells of a 384-well plate.
Inhibitor compounds were added immediately thereafter to bring the
final well volume to 40 .mu.l, and the plates were incubated for 4
h at 37.degree. C. Compounds were tested individually and in
combination at a fixed molar ratio or mass ratio over a range of
serial dilutions. The plates were then read on a fluorescence plate
reader (Victor2, Perkin Elmer, Boston, Mass.) using the
excitation/emission filter combinations shown in Table 6.
TABLE-US-00004 TABLE 6 Excitation/emission filter combinations for
RET assay Scan No. Excitation wavelength Emission wavelength 1 450
nm/50 nm 530 nm/25 nm 2 530 nm/25 nm 590 nm/35 nm 3 450 nm/50 nm
590 nm/35 nm
[0162] The "% RET" was calculated according to the following
formula after subtraction of background (blank) readings:
%
RET=100.times.[(A.sub.3-(A.sub.1.times.F.sub.spill)-(A.sub.2.times.R.s-
ub.spill))/A.sub.2]
Where:
[0163] F.sub.spill=HeLa cells alone, Scan 3/Scan 1; [0164]
R.sub.spill=CEM cells alone, Scan 3/Scan 2; [0165] A.sub.1=Scan 1
value for HeLa and CEM cells in combination; [0166] A.sub.2=Scan 2
value for HeLa and CEM cells in combination; and [0167]
A.sub.3=Scan 3 value for HeLa and CEM cells in combination.
[0168] The "% Inhibition" was calculated according to the following
formula:
% Inhibition=100.times.[(Max % RET-% RET for sample well)/(Max %
RET-Min % RET)]
Where:
[0169] Max % RET=average of % RET values for HeLa and CEM cell
combination without added inhibitor; and [0170] Min % RET=average
of % RET values for HeLa and CEM cell combination in presence of
500 ng/ml of Leu-3a mAb (an antibody that targets CD4 and fully
blocks fusion in the RET assay at this concentration).
[0171] Fifty percent inhibition (IC.sub.50) values were determined
by fitting the inhibition data with a non-linear, four-parameter,
variable slope equation (GraphPad Prism, ver. 4.02; GraphPad
Software, San Diego, Calif.). Upper and lower inhibition values
were constrained to 100% and 0%, respectively for curve
fitting.
Synergy Determinations
[0172] Cooperative inhibition effects of drug combinations were
determined by the method of Chou and Talalay (1984). IC.sub.50
values were generated for all combinations as described above.
Combination Index (CI) and Dose Reduction (DR) values were
calculated according to the following formulas:
C l = ( I C 50 D comb 1 I C 50 D solo 1 ) + ( I C 50 D comb 2 I C
50 D solo 2 ) + .alpha. ( ( I C 50 D comb 1 ) ( I C 50 D comb 2 ) (
I C 50 D solo 1 ) ( I C 50 D solo 2 ) ) ##EQU00001## DR (for
compound 1)=(IC.sub.50 Dsolo1/IC.sub.50 Dcomb1)
DR (for compound 2)=(IC.sub.50 Dsolo2/IC.sub.50 Dcomb2)
Where:
[0173] "IC.sub.50 Dcomb1"=IC.sub.50 of drug 1 in combination with
drug 2; [0174] "IC.sub.50 Dsolo1"=IC.sub.50 of drug 1 when tested
alone; [0175] "IC.sub.50 Dcomb2"=IC.sub.50 of drug 2 in combination
with drug 1; [0176] "IC.sub.50 Dsolo2"=IC.sub.50 of drug 2 when
tested alone; [0177] .alpha.=0 if the effects of the two drugs are
mutually exclusive; and [0178] .alpha.=1 if the effects of the two
drugs are mutually nonexclusive
[0179] Combinations with CI<1 are determined to be synergistic,
whereas combinations with CI>1 are determined to be
antagonistic. Additivity is reflected in combinations for which
CI=1.
[0180] Ninety five percent Confidence Intervals were calculated in
Microsoft Excel using the formula:
=Confidence(alpha,stdev,n)
Where:
[0181] alpha=0.05 (95% confidence); [0182] stdev=standard deviation
of dataset mean; and [0183] n=number of replicates.
Results
Preparation of Small-Molecule Fusion Inhibitors
[0184] SCH-D, TAK-779, UK427,857, and BMS378806 were prepared by
commercial sources. The desired quantities and HPLC purity of the
compounds were realized. Purity of the compounds was supported by
results obtained from elemental analysis, and the identities of the
products were confirmed by proton NMR (proton and carbon-13) and/or
mass spectrum data.
Synergistic Interactions Revealed by RET Assay
[0185] Synergy experiments were conducted using the cell-cell RET
fusion assay to assess initially the potential for cooperative
interactions between PRO 140 and small-molecule and peptide-based
inhibitors of CCR5, CD4, HIV-1 gp120 and HIV-1 gp41. The
experiments were then extended to the CCR5-specific murine
monoclonal antibody, 2D7 (Wu et al., 1997).
[0186] Experiments measuring inhibition of HIV-1 Env-mediated
fusion were first conducted using combinations of PRO 140 with,
respectively, PRO 140 itself, 3 small-molecule CCR5 antagonists
(SCH-D, TAK-779, UK427857), the natural peptide ligand of CCR5
(RANTES), and an anti-CCR5 mAb (2D7), a peptide-based inhibitor of
gp41 (T-20), a protein-based inhibitor of gp120 (PRO 542), a
small-molecule inhibitor of gp120 (BMS378806), and an anti-CD4 mAb
(Leu3A). Mass ratios of PRO 140 to other entry inhibitors ranged
from 0.75 to 364. The results are shown in Table 7.
TABLE-US-00005 TABLE 7 Combination Index and Dose Reduction Values
for inhibition of HIV-1 Env-mediated fusion with combinations of
PRO 140 and entry inhibitors Mean Dose Mean Dose PRO 140 in
Reduction Reduction (Cpd combination No. of Cpd mass Inhibitor Mean
CI.sup.c (PRO 140) in combination) with:.sup.a tests ratios.sup.b
target Cell-cell fusion assay PRO 140 9 1 CCR5 0.97 .+-. 0.08 2.07
.+-. 0.18 2.07 .+-. 0.18 TAK-779 8 282 CCR5 0.36 .+-. 0.10 4.10
.+-. 2.03 15.86 .+-. 7.10 SCH-D 9 279 CCR5 0.51 .+-. 0.05 4.21 .+-.
0.96 3.90 .+-. 0.71 UK-427,857 3 292 CCR5 0.59 .+-. 0.04 4.16 .+-.
0.41 2.98 .+-. 0.65 RANTES 4 19 CCR5 0.59 .+-. 0.08 4.13 .+-. 0.99
3.24 .+-. 1.06 2D7 2 1 CCR5 0.93 .+-. 0.04 1.87 .+-. 0.07 2.54 .+-.
0.13 T-20 7 33 gp41 0.84 .+-. 0.16 1.77 .+-. 0.40 7.47 .+-. 3.34
PRO 542 6 0.75 gp120 0.96 .+-. 0.17 1.59 .+-. 0.21 5.54 .+-. 1.49
BMS-378806 7 364 gp120 1.21 .+-. 0.21 1.64 .+-. 0.30 2.85 .+-. 0.76
.sup.aCompounds were tested at a 1:1 molar ratio. .sup.bMass of PRO
140/mass of other HIV-1 entry inhibitor tested in combination.
Molecular weights of inhibitors are: PRO 140 .apprxeq. 150,000
g/mole; SCH-D = 538 g/mole; TAK-779 = 531 g/mole (hydrochloride
salt); UK-427,857 = 514 g/mole; RANTES .apprxeq. 7,800 g/mole; 2D7
.apprxeq. 150,000 g/mole; T-20 = 4,492 g/mole; PRO 542 .apprxeq.
200,000 g/mole; BMS-378806 = 412 g/mole. .sup.cCombination Index at
IC.sub.50 value. The mutually exclusive CI formula (.alpha. = 0)
was utilized for PRO 140 in combination with molecules that bind
CCR5, and the mutually non-exclusive formula (.alpha. = 1) was
utilized for PRO 140 in combination with molecules that bind other
targets (Chou and Rideout, 1991).
[0187] Two small-molecule CCR5 antagonists, SCH-D and TAK-779, were
assayed in combination. PRO 542, a recombinant antibody-like fusion
protein in which the heavy- and light-chain variable domains of
human IgG2 have been replaced with the D1D2 domains of human CD4,
was also tested in combination with the anti-CD4 mAb, Leu-3A. The
results of these assays are shown in Table 8.
TABLE-US-00006 TABLE 8 Other drug combinations tested in the RET
assay for cooperativity Molar ratios Mean CI .+-. Mean DR Mean DR
Drug 1 Drug 2 (Drug 1 to 2) N stdev.sup.a (Drug 1) (Drug 2) SCH-D
TAK-779 1:1 4.sup.b 1.12 .+-. 0.32 1.48 .+-. 0.96 4.31 .+-. 1.82
PRO 542 Leu-3A 22.9:1 2.sup. 16.9 .+-. 0.3 0.7 .+-. 0 0.16 .+-. 0
.sup.aCI values were calculated using the mutually exclusive
formula for SCH-D vs. TAK-779 (i.e., where .alpha. = 0) and the
mutually non-exclusive formula for PRO 542 vs. Leu-3A (i.e., where
.alpha. = 1; see methods). .sup.bOne aberrant datapoint was culled
from the calculation of Mean CI and Mean DRs.
[0188] The effect of varying the relative amounts of compounds in
the combinations on the level of cooperativity was also measured.
Molar ratios of 5:1 and 1:5 PRO 140 were used. The results are
tabulated in Table 9, and the mean CI values with 95% confidence
intervals are plotted in FIG. 4 for the 1:1 molar ratio data. In
addition to PRO 140, the inhibitory activity of mAb 2D7, a
CCR5-specific murine antibody (Wu et al., 1997) was also tested in
combination with the small-molecule CCR5 antagonists and with
RANTES using the fluorescent RET assay. The results are shown in
Table 10.
TABLE-US-00007 TABLE 9 Combination Index and Dose Reduction Values
for inhibition of HIV-1 Env-mediated fusion with combinations of
PRO 140 and entry inhibitors Mean Mean Dose PRO 140 in Combination
Reduction Mean Dose Reduction combination Cpd Mass Index.sup.c (PRO
140) (Cpd. in combination) with: Ratio.sup.a Ratios.sup.b Cell-cell
fusion assay PRO 140 5:1 5 1.15 .+-. 0.09 1.05 .+-. 0.08 5.26 .+-.
0.41 PRO 140 1:5 0.2 1.09 .+-. 0.08 5.54 .+-. 0.38 1.10 .+-. 0.08
TAK-779 5:1 1410 0.57 .+-. 0.07 1.89 .+-. 0.14 33.59 .+-. 18.85
TAK-779 1:5 56.4 0.52 .+-. 0.20 5.58 .+-. 0.52 3.78 .+-. 1.95 SCH-D
5:1 1395 0.66 .+-. 0.10 1.92 .+-. 0.40 8.44 .+-. 1.27 SCH-D 1:5
55.8 0.69 .+-. 0.05 9.95 .+-. 2.03 1.73 .+-. 0.19 UK-427,857 5:1
1460 0.66 .+-. 0.11 2.00 .+-. 0.35 7.25 .+-. 2.19 UK-427,857 1:5
58.4 0.73 .+-. 0.05 11.31 .+-. 2.14 1.58 .+-. 0.17 RANTES 5:1 95
0.84 .+-. 0.14 1.63 .+-. 0.43 5.39 .+-. 1.13 RANTES 1:5 3.8 0.66
.+-. 0.06 13.64 .+-. 4.75 1.75 .+-. 0.28 T-20 5:1 165 1.10 .+-.
0.12 0.98 .+-. 0.11 31.85 .+-. 10.19 T-20 1:5 6.6 0.76 .+-. 0.27
2.93 .+-. 0.68 3.85 .+-. 1.50 PRO 542 5:1 3.75 1.13 .+-. 0.10 1.01
.+-. 0.07 15.73 .+-. 4.15 PRO 542 1:5 0.15 1.18 .+-. 0.17 2.83 .+-.
0.50 1.71 .+-. 0.29 BMS-378806 5:1 1820 1.12 .+-. 0.10 1.14 .+-.
0.06 8.88 .+-. 4.16 BMS-378806 1:5 72.8 1.55 .+-. 0.24 3.64 .+-.
0.73 1.07 .+-. 0.31 .sup.aMolar ratio of PRO 140 to other entry
inhibitor tested in combination (n = 3 for all experimental
results) .sup.bMass of PRO 140/mass of other HIV-1 entry inhibitor
tested in combination. Molecular weights of inhibitors are: PRO 140
.apprxeq. 150,000 g/mole; SCH-D = 538 g/mole; TAK-779 = 531 g/mole
(hydrochloride salt); UK-427,857 = 514 g/mole; RANTES .apprxeq.
7,800 g/mole; T-20 = 4,492 g/mole; PRO 542 .apprxeq. 200,000
g/mole; BMS-378806 = 412 g/mole. .sup.cCombination Index at
IC.sub.50 value. The mutually exclusive CI formula (.alpha. = 0)
was utilized for PRO 140 in combination with molecules that bind
CCR5, and the mutually non-exclusive formula (.alpha. = 1) was
utilized for PRO 140 in combination with molecules that bind other
targets (Chou and Rideout, 1991).
TABLE-US-00008 TABLE 10 Combination Index and Dose Reduction Values
for inhibition of HIV-1 Env-mediated fusion with combinations of
2D7 and entry inhibitors Mean Mean Dose Mean Dose 2D7 in
Combination Reduction Reduction (Cpd combination Cpd Mass Inhibitor
Index.sup.b (2D7) in combination) with:.sup.a Ratios.sup.c target
Cell-cell fusion assay TAK-779 282 CCR5 0.15 .+-. 0.03 17.20 .+-.
3.23 11.95 .+-. 4.94 SCH-D 279 CCR5 0.57 .+-. 0.10 3.25 .+-. 0.56
4.04 .+-. 0.78 UK427857 292 CCR5 0.58 .+-. 0.03 2.45 .+-. 0.12 5.73
.+-. 0.54 RANTES 19 CCR5 0.62 .+-. 0.04 1.94 .+-. 0.08 10.18 .+-.
1.86 PRO 140 1 CCR5 0.93 .+-. 0.04 2.54 .+-. 0.13 1.87 .+-. 0.07
.sup.aCompounds were tested at a 1:1 molar ratio (all data are n =
3 except for 2D7 and PRO 140, where n = 2) .sup.bCombination Index
at IC.sub.50 value. The mutually exclusive CI formula (.alpha. = 0)
was utilized for 2D7 in combination with molecules that bind CCR5
(Chou and Rideout, 1991). .sup.cMass of 2D7/mass of other HIV-1
entry inhibitor tested in combination. Molecular weights of
inhibitors are: 2D7 .apprxeq. 150,000 g/mole; SCH-D = 538 g/mole;
TAK-779 = 531 g/mole (hydrochloride salt); UK-427,857 = 514 g/mole;
RANTES .apprxeq. 7,800 g/mole.
Example 2
Combination Testing of PRO 140 with Small Molecule, Peptide and
Protein Inhibitors, and HIV-1 in the HIV-1 Pseudovirus Particle
(HIV-1pp) Assay
Materials and Methods
Preparation of HIV-1 Pseudoparticles
[0189] HIV-1 pseudoparticles (HIV-1pp) are generated in 293T cells
by transient coexpression of an HIV-1-based NL4/3luc+env- plasmid
and a construct encoding HIV-1.sub.JRFL Env. The NL4/3luc+env-
plasmid was obtained from the NIH AIDS Research and Reference
Reagent Program (Cat. No. 3418), and the HIV-1.sub.JRFL Env was
inserted into the pcDNA3.1 vector (Invitrogen). Briefly, 293T cells
are calcium phosphate transfected with a 1:1 ratio of NL4/3luc+env-
reporter vector and Env expression vector in Hepes buffer
(Profection Mammalian Transfection Kit, Promega). After 16 h the
transfection medium is aspirated and fresh cell culture medium
(DMEM with 10% FBS, glutamine and antibiotics) is added and the
incubation is continued at 37.degree. C. for an additional 24-32 h.
Cell culture supernatants are collected 48 h post-transfection and
centrifuged at 1,400 rpm for 10 min to pellet cell debris. The
viral supernatant is brought to a final concentration of 5% sucrose
and stored aliquoted at -80.degree. C.
Cells
[0190] U87-CD4-CCR5 cells were obtained from the NIH AIDS Research
and Reference Program (Cat. No. 4035). These cells are maintained
in culture medium (DMEM with 10% FBS, antibiotics and glutamine)
containing 0.3 mg/ml G418 and 0.5 mg/ml puromycin. Cells are grown
in T175-cm.sup.2 flasks at 37.degree. C. and diluted 1:5 every 3-4
days. For assay plate preparation, cells are trypsinized and seeded
into wells of 96-well tissue-culture treated flat bottom opaque
polystyrene plates (Perkin Elmer, Boston, Mass.) at a density of
3.times.10.sup.3 cells/well. Plates are incubated for no more than
4 h at 37.degree. C. in a humidified 5% CO.sub.2 incubator prior to
their use in the HIV-1 pp susceptibility assay.
Compound Preparation
[0191] Fifty .mu.l of diluted compound at 4.times. the desired
final concentration are added per well. For compounds solubilized
in DMSO, the 4.times. stock will contain 2% DMSO (such that the
final DMSO concentration in the assay is always 0.5% for small
molecules). Control wells receiving no compound are included on
each plate. In addition, an AZT inhibition control is included in
each assay. Compounds are tested individually and at a fixed mass
or molar ratio over a broad range of concentrations.
Virus Addition
[0192] A vial of frozen, aliquoted HIV-1pp is thawed in a
37.degree. C. waterbath and then placed on wet ice. Virus is
diluted in cold cell culture medium as necessary to achieve the
desired final virus concentration in the HIV-1pp assay (about
10,000 relative light units (rlu) per well). 50 .mu.l of diluted
virus are added per well, bringing the final well volume to 200
.mu.l. A no-virus control (minimum or background luminescence) and
a no-compound control (maximum luminescence) are included on each
plate. The plates are incubated for 72 h at 37.degree. C. in a
humidified 5% CO.sub.2 incubator followed by processing for
luciferase signal (see below).
Plate Processing for Luciferase Assay
[0193] Assay medium is aspirated and 200 .mu.l of PBS are added to
each well. The PBS is aspirated and 50 .mu.l of 1.times. Cell Lysis
Reagent (Promega--Cat. No. E1531) are added to each well. Assay
plates are then frozen for at least 2 h at -80.degree. C. followed
by thawing at room temperature and vigorous mixing with an
electronic pipettor. 25 .mu.l from each well are transferred to an
opaque 96-well plate (Costar #3922). Four replicates are pooled
into the same well on the opaque plate. 100 .mu.l of freshly thawed
and reconstituted luciferase substrate (Luciferase Assay System,
Promega--Cat. No. E1501) are added to each well of the plate with
the electronic pipettor, and luminescence is detected immediately
on a Dynex MLX plate reader set to medium gain.
Data Analysis
[0194] Neutralization activity is displayed by plotting the percent
inhibition of luciferase activity (after background rlu values are
subtracted from all datapoints) versus log.sub.10 drug
concentration. The percent inhibition is derived as follows:
[1-(luciferase activity in the presence of drug/luciferase activity
in the absence of drug)].times.100. IC.sub.50 values are determined
by fitting the inhibition data with a non-linear, four-parameter,
variable slope equation (GraphPad Prism, ver. 4.02; GraphPad
Software, San Diego, Calif.). Upper and lower inhibition values are
constrained to 100% and 0%, respectively for curve fitting.
Synergy Determination
[0195] Cooperative interactions between PRO 140 and small-molecule
and peptide-based inhibitors of CCR5, CD4, HIV-1 gp120, HIV-1 gp41
and HIV-1 reverse transcriptase (see Tables 4 and for listing of
HIV-1 inhibitors approved for clinical use) are determined as
described in Example 1. Cooperative inhibition effects of drug
combinations are determined by the method of Chou and Talalay
(1984). IC.sub.50 values are generated for all combinations as
described above. Combination Index (CI) and Dose Reduction (DR)
values are calculated according to the following formulas:
C l = ( I C 50 D comb 1 I C 50 D solo 1 ) + ( I C 50 D comb 2 I C
50 D solo 2 ) + .alpha. ( ( I C 50 D comb 1 ) ( I C 50 D comb 2 ) (
I C 50 D solo 1 ) ( I C 50 D solo 2 ) ) ##EQU00002## DR (for
compound 1)=(IC.sub.50 Dsolo1/IC.sub.50 Dcomb1)
DR (for compound 2)=(IC.sub.50 Dsolo2/IC.sub.50 Dcomb2)
Where:
[0196] "IC.sub.50 Dcomb1"=IC.sub.50 of drug 1 in combination with
drug 2; [0197] "IC.sub.50 Dsolo1"=IC.sub.50 of drug 1 when tested
alone; [0198] "IC.sub.50 Dcomb2"=IC.sub.50 of drug 2 in combination
with drug 1; [0199] "IC.sub.50 Dsolo2"=IC.sub.50 of drug 2 when
tested alone; [0200] .alpha.=0 if the effects of the two drugs are
mutually exclusive; and [0201] .alpha.=1 if the effects of the two
drugs are mutually nonexclusive.
[0202] Combinations with CI<1 are determined to be synergistic,
whereas combinations with CI>1 are determined to be
antagonistic. Additivity is reflected in combinations for which
CI=1.
Example 3
Combination Testing of PRO 140 with Small Molecule, Peptide and
Protein Inhibitors in the HIV-1 Authentic Virus Replication
Assay
Materials and Methods
Preparation of PBMCs
[0203] Replication of authentic HIV-1 is measured in activated
peripheral blood mononuclear cells (PBMCs) using the
monocyte/macrophage-tropic HIV-1 clone, JRFL (HIV-1.sub.JRFL), for
these studies.
[0204] PBMCs are isolated from 4 separate donors (Leukopacks) by
centrifugation on a Ficoll gradient. CD8 cells are depleted using
RosetteSep CD8 Depletion Cocktail (#15663, StemCell Research,
Vancouver, BC). Cells are diluted to 4.times.10.sup.6/ml and added
in equal parts to three T175-cm.sup.2 flasks and then stimulated by
addition of one of the following media: IL-2 Medium [RPMI 1640
(#10-040-CV, Cellgro, Herndon, Va.), 10% FBS (#35-010-CV), 2 mM
L-Glutamine (#25-005-CI), 100 U/ml IL-2 (Sigma, St. Louis, Mo.)];
PHA 5 Medium: [IL-2 Medium with 5 ug/ml Phytohemagglutinin PHA-P
(PHA) (#L8754, Sigma, St. Louis, Mo.), filtered]; or PHA 0.5
Medium: [IL-2 Medium with 0.5 ug/ml PHA, filtered]. Each flask
receives a total of 50-150 ml of medium. Flasks are incubated for 3
days at 37.degree. C. followed by pooling of the contents prior to
use in the infection assay.
Virus Titration
[0205] Serial dilutions of virus are tested in quadruplicate on
activated PBMCs (1.4.times.10.sup.5 PBMC/well). Titration Medium
[IL-2 Medium with 100 IU/ml penicillin/streptomycin (#30-002-CI,
Cellgro)] is utilized for virus titrations. Fifty .mu.l of diluted
virus is added to 100 .mu.l of PBMCs in flat bottom, tissue-culture
treated 96-well plates (VWR# 29442-054, Corning, Corning, N.Y.) and
the plates are incubated at 37.degree. C. in a humidified, 5%
CO.sub.2 incubator. After 7 days, 50 .mu.l are removed from each
well and tested for virus levels by p24 antigen ELISA (Perkin
Elmer, Boston, Mass.). Virus titer is determined by the method of
Reed and Muench (Table 11).
Neutralization Assay
[0206] Stimulated PBMCs are seeded into wells of 96-well flat
bottom plates at a density of 1.4.times.10.sup.5 cells/well. Virus
is diluted to 2,000 TCID.sub.50/ml and mixed with serial 0.5
log.sub.10 dilutions of compound for 1 h at 37.degree. C. prior to
addition to the cell plates. The final amount of virus added per
well is 100 TCID.sub.50. The final DMSO concentration in the assay
is always 0.5% whenever small molecule inhibitors are being tested.
Plates are incubated at 37.degree. C. for 5 days, at which time an
aliquot of supernatant is removed for p24 antigen ELISA. If control
wells (virus without inhibitor) exhibit low p24 antigen levels then
the plates are brought back to full volume with Titration medium
and incubated for an additional 24 h.
TABLE-US-00009 TABLE 11 Reed and Muench formula for calculating
virus titer.sup.a No. of pos. TCID.sub.50/ml wells (10.sup.x) 1
0.74 2 0.83 3 0.92 4 1.00 5 1.09 6 1.17 7 1.26 8 1.35 9 1.44 10
1.52 11 1.61 12 1.70 13 1.79 14 1.87 15 1.96 16 2.05 17 2.14 18
2.22 19 2.31 20 2.40 21 2.49 22 2.57 23 2.66 24 2.75 25 2.83 26
2.92 27 3.01 28 3.10 29 3.18 30 3.27 31 3.36 32 3.45 33 3.53 34
3.62 35 3.71 36 3.80 37 3.88 38 3.97 39 4.06 40 4.15 41 4.23 42
4.32 43 4.41 44 4.49 45 4.58 46 4.67 47 4.76 48 4.84 49 4.93 50
5.02 51 5.11 52 5.19 53 5.28 54 5.37 55 5.46 56 5.54 57 5.63 58
5.72 59 5.81 60 5.89 61 5.98 62 6.07 63 6.15 64 6.24 65 6.33 66
6.42 67 6.50 68 6.59 69 6.68 70 6.77 71 6.85 72 6.94 73 7.03 74
7.12 75 7.20 76 7.29 77 7.38 78 7.47 79 7.55 80 7.64 .sup.aTo
calculate virus titer, first multiply the total number of positive
wells by 2 (the chart was designed to be used with replicates of
8), then look up the corresponding TCID.sub.50/mL titer and add 0.7
(the formula requires the addition of a log dilution factor).
Data Analysis
[0207] Neutralization activity is displayed by plotting the percent
inhibition of p24 antigen production (after background values are
subtracted from all datapoints) versus log.sub.10 drug
concentration. The percent inhibition is derived as follows [1-(p24
levels in the presence of drug/p24 levels in the absence of
drug)].times.100. IC.sub.50 values are determined by fitting the
inhibition data with a non-linear, four-parameter, variable slope
equation (GraphPad Prism, ver. 4.02; GraphPad Software, San Diego,
Calif.). Upper and lower inhibition values are constrained to 100%
and 0%, respectively for curve fitting.
Synergy Determinations
[0208] Cooperative interactions between PRO 140 and small-molecule
and peptide-based inhibitors of CCR5, CD4, HIV-1 gp120, HIV-1 gp41,
HIV-1 reverse transcriptase and HIV-1 protease (Table 8) are
determined as described for Example 1. Cooperative inhibition
effects of drug combinations are determined by the method of Chou
and Talalay (1984). IC.sub.50 values are generated for all
combinations as described above. Combination Index (CI) and Dose
Reduction (DR) values are calculated according to the following
formulas:
C l = ( I C 50 D comb 1 I C 50 D solo 1 ) + ( I C 50 D comb 2 I C
50 D solo 2 ) + .alpha. ( ( I C 50 D comb 1 ) ( I C 50 D comb 2 ) (
I C 50 D solo 1 ) ( I C 50 D solo 2 ) ) ##EQU00003## DR (for
compound 1)=(IC.sub.50 Dsolo1/IC.sub.50 Dcomb1)
DR (for compound 2)=(IC.sub.50 Dsolo2/IC.sub.50 Dcomb2)
Where:
[0209] "IC.sub.50 Dcomb1"=IC.sub.50 of drug 1 in combination with
drug 2; [0210] "IC.sub.50 Dsolo1"=IC.sub.50 of drug 1 when tested
alone; [0211] "IC.sub.50 Dcomb2"=IC.sub.50 of drug 2 in combination
with drug 1; [0212] "IC.sub.50 Dsolo2"=IC.sub.50 of drug 2 when
tested alone; [0213] .alpha.=0 if the effects of the two drugs are
mutually exclusive; and [0214] .alpha.=1 if the effects of the two
drugs are mutually nonexclusive.
[0215] Combinations with CI<1 are determined to be synergistic,
whereas combinations with CI>1 are determined to be
antagonistic. Additivity is reflected in combinations for which
CI=1.
Discussion
[0216] PRO 140 is a CCR5-specific mAb being developed for HIV-1
therapy. It is a humanized IgG4,.kappa. version (see PCT
International Publication No. WO 03/072766, published Sep. 4, 2003)
of the murine antibody, PA14 (Olson et al., 1999; PCT International
Publication No. WO 00/35409, published Jun. 20, 2000), which binds
to the CCR5 receptor on the surface of a cell and inhibits
CCR5-mediated fusion of HIV-1 to the cell. The studies described
herein concern the testing of the antiviral activity of PRO 140 in
combination with small-molecule and peptide inhibitors of HIV-1
infection. Data generated from this testing were analyzed for
potential cooperative effects on inhibition of HIV-1 infection.
[0217] In one series of experiments, inhibition of HIV-1 infection
was assayed using a fluorescence resonance energy transfer (RET)
assay, which measures the fusion of effector cells
(HeLa-Env.sub.JRFL) expressing recombinant HIV-1 strain JRFL
envelope glycoproteins (Env) to target cells (CEM NKR-CCR5)
expressing CD4 and CCR5 (Litwin et al., 1996). In this assay,
effector cells are labeled with the F18 dye and target cells with
the R18 dye. HIV-1 Env-mediated fusion of effector and target cells
results in the placement of these two dyes within close proximity
in the cell membrane. When F18 is excited at its optimum wavelength
(450 nm), it emits light at a wavelength (530 nm) that will excite
R18 when the two dyes are co-localized in the same membrane,
resulting in R18-specific emission at 590 nm. Drug susceptibility
is measured by adding serial concentrations of drugs to target
cells prior to addition of effector cells. Inhibition of HIV-1
Env-mediated fusion is reflected in a reduction in fluorescence
emission due to R18 in a dose-dependent manner, providing a
quantitative measure of drug activity.
[0218] Initial experiments measuring inhibition of HIV-1
Env-mediated fusion were conducted in order to demonstrate the
robustness of the assay system for quantifying cooperative
interactions. In these experiments, PRO 140 was run in combination
with itself, a combination that should result in combination index
(CI) values indicative of additive interactions. Using the
methodology of Chou and Talalay (1984), CI values of <1.0, =1.0
and >1.0 are taken to indicate synergistic, additive and
antagonistic interactions, respectively. Indeed, PRO 140 run in
combination with itself returned a CI value of 0.97.+-.0.08 (n=9;
Table 7), indicating that the assay system accurately represented
this interaction.
[0219] Synergy experiments were then conducted between PRO 140 and
3 small-molecule (SCH-D, TAK-779, UK427857), one peptide (RANTES)
and one mAb (2D7) antagonist of CCR5. In addition, cooperative
interactions were measured between PRO 140 and T-20 (peptide-based
inhibitor of gp41), PRO 542 (protein-based inhibitor of gp120),
BMS378806 (small molecule inhibitor of gp120) and Leu-3A (anti-CD4
mAb).
[0220] The results (see Table 7) revealed potent synergy between
PRO 140 and all 3 small-molecule CCR5 antagonists as well as
RANTES. CI values between PRO 140 and these CCR5 antagonists ranged
from 0.36.+-.0.10 to 0.59.+-.0.08. Dose reduction values indicated
that the compound in combination exerted about a 4-fold effect on
PRO 140 activity, whereas the effect of PRO 140 on the compound in
combination ranged from about 3- to about 16-fold (Table 7). Modest
synergy to additivity was observed between PRO 140 and T-20, PRO
542, BMS-378806 and 2D7 (CI=0.84.+-.0.16, 0.96.+-.0.17,
1.21.+-.0.21, and 0.93.+-.0.04, respectively).
[0221] Small molecule antagonists of CCR5 run in combination (SCH-D
and TAK-779) returned a mean CI value of 1.12.+-.0.32, indicating a
slightly additive interaction (Table 8). Conversely, the
combination of the recombinant antibody-like fusion protein PRO 542
with the anti-CD4 mAb, Leu-3A, resulted in a mean CI value of
16.9.+-.0.3, indicating potent antagonism between these two HIV-1
inhibitors (Table 8).
[0222] Varying the molar ratios of compounds demonstrated similar
patterns of cooperativity. At both 5:1 and 1:5 molar ratios of PRO
140 to SCH-D, TAK-779, UK-427,857 and RANTES, potent synergistic
inhibition of HIV-1-Env-mediated entry was observed (Table 9). This
represents a broad range of inhibitor mass ratios, from a low of
0.15 to a high of 1,820. CI values between PRO 140 and CCR5
antagonists ranged from 0.52.+-.0.20 to 0.84.+-.0.14. More modest
synergy to additivity was observed for combinations of PRO 140 with
T-20, PRO 542 or BMS-378806. The results of these investigations
identify clearly the potent synergistic activities of PRO 140 with
CCR5 antagonists, as well as more modest synergy between PRO 140
and T-20 (see FIG. 4).
[0223] The HIV-1 inhibitory activity of the CCR5-specific murine
mAb, 2D7, in combination with the small-molecule CCR5 antagonists
and with RANTES, was also tested using the fluorescent RET assay.
2D7 was found to act synergistically with these CCR5 antagonists
and with RANTES (Table 10). CI values between 2D7 and these CCR5
antagonists ranged from 0.15.+-.0.03 to 0.62.+-.0.04. Dose
reduction values indicated that the compound in combination exerted
about a 2- to 3-fold effect on 2D7 activity, except for TAK-779
which had an approximately 17-fold effect on 2D7 activity. The
effect of 2D7 on the compound in combination ranged from about 2-
to about 12-fold (Table 10). As observed previously, PRO 140 and
2D7 in combination were essentially additive or modestly
synergistic (CI=0.93.+-.0.04).
[0224] These results indicate that synergistic inhibition of HIV-1
Env-mediated cell-cell fusion is observed between multiple mAbs and
small molecules that bind to CCR5. This property may be broadly
applicable to mAbs that target CCR5, including, for example, the
mAb CCR5 mAb004 that has been shown to bind to and antagonize CCR5
and block HIV-1 entry in a cell-cell fusion assay (Roschke et al.,
2004). A large and growing number of small molecules have been
identified as CCR5 antagonists (see Table 12). Certain of these
small molecule CCR5 antagonists may also produce synergistic
inhibition of HIV-1 Env-mediated fusion in combination with PRO 140
and other anti-CCR5 mAbs.
[0225] An alternative approach for examining synergistic
interactions utilizes a virus-cell fusion assay as described
previously (Nagashima et al., 2001; Trkola et al., 1998). In this
assay an HIV genomic vector (pNLluc.sup.+Env.sup.-) containing a
luciferase reporter gene is pseudotyped with Env from
HIV-1.sub.JRFL. Recombinant pseudotyped virus particles are used to
infect U87 cells expressing CD4 and CCR5 (U87-CD4-CCR5). Production
of luciferase in target cells is dependent on virus entry and the
completion of one round of virus replication. Drug susceptibility
is measured by adding serial concentrations of drugs to target
cells prior to addition of pseudotyped virus particles. Inhibition
of virus entry is reflected in a reduction in luciferase activity
in a dose-dependent manner, providing a quantitative measure of
drug susceptibility. Since the HIV genomic vector requires
expression of functional HIV-1 reverse transcriptase (RT) to drive
luciferase expression, this pseudovirus assay is also sensitive to
inhibition by nucleotide/nucleoside reverse transcriptase
inhibitors (NRTIs) and non-nucleoside reverse transcriptase
inhibitors (NNRTIs). As such, the HIV-1pp assay is suitable for
examining cooperative interactions between PRO 140 and
small-molecule, peptide and protein inhibitors of CCR5, CD4, HIV-1
gp120, HIV-1 gp41 and HIV-1 reverse transcriptase.
TABLE-US-00010 TABLE 12 Small-Molecule CCR5 antagonists
Small-Molecule CCR5 antagonist Reference 1,3,4-trisubstituted
pyrrolidines Kim et al., 2005 Modified
4-piperidinyl-2-phenyl-1-(phenylsulfonylamino)- Shah et al., 2005
butanes Anibamine.TFA, Ophiobolin C, and 19,20-epoxycytochalasin Q
Jayasuriya et al., 2004 5-(piperidin-1-yl)-3-phenyl-pentylsulfones
Shankaran et al., 2004a
4-(heteroarylpiperdin-1-yl-methyl)-pyrrolidin-1-yl-acetic acid
Shankaran et al., 2004b antagonists Agents containing
4-(pyrazolyl)piperidine side chains Shu et al., 2004 Agents
containing 4-(pyrazolyl)piperidine side chains. Shen et al., 2004a;
2004b 3-(pyrrolidin-1-yl)propionic acid analogues Lynch et al.,
2003c [2-(R)-[N-methyl-N-(1-(R)-3-(S)-((4-(3-benzyl-1-ethyl-(1H)-
Kumar et al., 2003 pyrazol-5-yl)piperidin-1-yl)methyl)-4-(S)-(3-
fluorophenyl)cyclopent-1-yl)amino]-3-methylbutanoic acid (MRK-1)]
1,3,4 trisubstituted pyrrolidines bearing 4-aminoheterocycle
Willoughby et al., 2003; Lynch substituted piperidine side chains
et al., 2003a; Lynch et al., 2003b; Hale et al., 2002 Bicyclic
isoxazolidines Lynch et al., 2002 Combinatorial synthesis of CCR5
antagonists Willoughby et al., 2001 Heterocycle-containing
compounds Kim et al., 2001b Antagonists containing hydantoins Kim
et al., 2001a 1,3,4 trisubstituted pyrrolidines Hale et al., 2001
1-[N-(methyl)-N-(phenylsulfonyl)amino]-2-(phenyl)-4-(4-(N- Finke et
al., 2001 (alkyl)-N-(benzyloxycarbonyl)
amino)piperidin-1-yl)butanes Compounds from the plant Lippia alva
Hedge et al., 2004 Piperazine-based CCR5 antagonists Tagat et al.,
2004 Oximino-piperidino-piperidine-based CCR5 antagonists Palani et
al., 2003b Rotamers of SCH 351125 Palani et al., 2003a
Piperazine-based symmetrical heteroaryl carboxamides McCombie et
al., 2003 Oximino-piperidino-piperidine Palani et al., 2002 amides
Sch-351125 and Sch-350634 Este, 2002 SCH-C Strizki et al., 2001
1-[(2,4-dimethyl-3-pyridinyl)carbonyl]-4-methyl-4-[3(S)- Tagat et
al., 2001a methyl-4-[1(S)-[4-(trifluoromethyl)phenyl]ethyl]-1-
piperazinyl]-piperidine N1-oxide (Sch-350634)
4-[(Z)-(4-bromophenyl)- Palani et al., 2001
(ethoxyimino)methyl]-1'-[(2,4-dimethyl-3-
pyridinyl)carbonyl]-4'-methyl-1,4'-bipiperidine N-oxide (SCH
351125) 2(S)-methyl piperazines Tagat et al., 2001b
Piperidine-4-carboxamide derivatives Imamura et al., 2005
1-benzazepine derivatives containing a sulfoxide moiety Seto et
al., 2005 anilide derivatives containing a pyridine N-oxide moiety
Seto et al., 2004a 1-benzothiepine 1,1-dioxide and 1-benzazepine
derivatives Seto et al., 2004b containing a tertiary amine moiety
N-[3-(4-benzylpiperidin-1-yl)propyl]-N,N'-diphenylureas Imamura et
al., 2004a 5-oxopyrrolidine-3-carboxamide derivatives Imamura et
al., 2004b Anilide derivatives with a quaternary ammonium moiety
Shiraishi et al., 2000 AK602/ONO4128/GW873140 Nakata et al., 2005
Spirodiketopiperazine derivatives Maeda et al., 2001; Maeda et al.,
2004 Selective CCR5 antagonists Thoma et al., 2004
[0226] A third approach for examining antiviral synergy utilizes a
whole virus assay. Cooperativity between all classes of inhibitor
molecules can be examined in this assay format.
[0227] In both the virus-cell fusion luciferase assay and the whole
virus assay, IC.sub.50 values are generated for all combinations as
described herein for the RET assay. Cooperative inhibition effects
of drug combinations are determined by the method of Chou and
Talalay (1984).
[0228] PRO 140 broadly and potently inhibited CCR5-mediated HIV-1
entry without CCR5 antagonism or other immunologic side effects in
preclinical testing. More recently, PRO 140 has demonstrated
favorable tolerability, PK and immunologic profiles in preliminary
results from an ongoing Phase 1a study in healthy volunteers. Thus,
in many respects, PRO 140 offers a novel and attractive product
profile for anti-HIV-1 therapy. Moreover, the activities of
anti-CCR5 mAbs are fundamentally distinct from, but complementary
to, those of small-molecule CCR5 antagonists (see Table 2).
[0229] It might have been expected that combinations of anti-CCR5
mAbs and non-antibody CCR5 antagonists would produce additive
effects in inhibiting fusion of HIV-1 to CD4.sup.+CCR5.sup.+ target
cells since both classes of agents bind to the same target
molecule. Surprisingly, however, the data presented herein reveal
that anti-CCR5 mAbs, exemplified by PRO 140 and 2D7, exhibited
potent and reproducible synergy with non-antibody CCR5 antagonists,
exemplified by SCH-D, TAK-779, UK-427,857 and RANTES, in inhibiting
HIV-1 Env-mediated cell-cell fusion. Synergies routinely translated
into 4- to 10-fold dose reductions, suggesting significant
improvement in inhibitory potency for the drug combinations. In
contrast, purely additive effects were observed for combinations of
non-antibody CCR5 antagonists. These findings likely reflect the
different patterns of CCR5 recognition of these molecules: whereas
small-molecule CCR5 antagonists bind a common hydrophobic pocket
within the transmembrane domains of CCR5, PRO 140 recognizes a
hydrophilic, extracellular epitope of CCR5. Overall, the data
support the use of PRO 140 in combination with non-antibody HIV-1
entry inhibitors and suggest that PRO 140 represents a distinct
subclass of CCR5 inhibitor.
[0230] Moreover, the available data suggest that the observed
synergy may also be exhibited by combinations involving anti-CCR5
mAbs other than PRO 140, including, but not limited to, mAb CCR5
mAb004 (Roschke et al., 2004), as well as non-antibody CCR5
antagonists other than SCH-D, TAK-779, UK-427,857 and RANTES. Thus,
these antibodies likely produce synergistic effects in combination
with GW873140 (Lalezari et al., 2004), TAK-652 (Baba et al., 2005),
and at least certain of the small-molecule CCR5 antagonists listed
in Table 12. Accordingly, combination therapy comprising
administration of anti-CCR5 mAbs and non-antibody CCR5 antagonists
may offer powerfully effective, new approaches to preventing and
treating HIV-1 infection. It is expected that such therapy will
result in more potent and more durable ant-HIV-1 treatments.
Additionally, the synergistic effects described herein may enable a
reduction in dosages of drugs administered to a subject as well as
a reduction in dosing frequency.
Example 4
Loading and Maintenance Dose Regimens
[0231] The loading regimen, which can, for example, be more
dose-intensive than the maintenance regimen, can, for example, have
the following characteristics:
[0232] Number of doses: 1 or more (up to about 5 doses).
[0233] Dose level: About 25%, 50%, 75%, 100%, 150% or 200% greater
than the maintenance dose regimen.
[0234] Dose frequency: About 1.5.times., 2.times., 3.times. or
4.times. more frequently than the maintenance dose regimen.
[0235] As an example, if the maintenance dose regimen is 2 mg/kg
every two weeks, the loading dose regimen could comprise weekly 2
mg/kg doses. Alternatively, the loading dose regimen could comprise
a single 4 mg/kg dose or multiple 4 mg/kg doses at weekly or
biweekly intervals.
[0236] The loading dose regimen can be designed, for example, so as
to accelerate the achievement of a pharmacokinetic steady state in
the subject, as defined by uniform peak and trough blood
concentrations of drug between doses. A preferred loading dose
regimen can be determined by routine experimentation wherein the
drug is administered to the subject by differing loading and
maintenance regimens, and blood levels of drug are measured.
[0237] Also, in another embodiment, PRO 140 is administered
according to a fixed-dose regimen such as, for example, 75 mg, 150
mg, 300 mg and 600 mg per administration.
Part III
Materials And Methods
Inhibitors
[0238] PRO 140 was expressed in mammalian cells and purified by
protein A, ion exchange and hydroxyapatite chromatographies.
UK427,857 (Dorr et al. 2005), SCH-D (Tagat et al. 2004), TAK-779
(Baba et al. 1999), enfuvirtide (T-20 (Wild et al. 1992);
BMS-378806 (Lin et al. 2003)) and PRO 542 (CD4-IgG2, (Allaway et
al. 1995)) were prepared according to published methods. Zidovudine
(azidothymidine, AZT), RANTES, the CCR5 mAb 2D7 and the CD4 mAb
Leu-3A were purchased from Sigma Chemicals (St. Louis, Mo.),
R&D Systems (Minneapolis, Minn.), Pharmingen (San Diego,
Calif.), and Becton Dickinson (Franklin Lakes, N.J.), respectively.
UK427,857 and SCH-D were radiolabeled with tritium by GE Healthcare
(Piscataway, N.J.), and PRO 140 was conjugated to phycoerythrin
(PE) by Southern Biotech, Inc. (Birmingham, Ala.).
HIV-1 Membrane Fusion Assay
[0239] HIV-1 envelope-mediated membrane fusion was examined using a
fluorescence resonance energy transfer (RET) assay (Litwin et al.
1996) with modifications. Briefly, HeLa cells that stably express
HIV-1.sub.JRFL gp120/gp41 (Litwin et al. 1996) and CEM.NKR-CCR5
cells (NIH AIDS Research and Reference Reagent Program,
(Spenlehauer et al. 2001; Trkola et al. 1999)) were labeled
separately overnight with fluorescein octadecyl ester (F18;
Molecular Probes, Eugene, Oreg.) and rhodamine octadecyl ester
(R18; Molecular Probes), respectively. Cells were washed in
phosphate-buffered saline containing 15% fetal bovine serum (PBSF)
and co-seeded at 15,000 cells/well into a 384-well plate.
Inhibitors were added, and the plates were incubated in PBSF plus
0.5% dimethlysulfoxide (DMSO) for 4 h at 37.degree. C. prior to
measurement of RET using a Victor.sup.2 plate reader (Perkin-Elmer,
Boston, Mass.) as previously described (Litwin et al. 1996). The
CD4 mAb Leu3a was used as a control inhibitor, and percent
inhibition was calculated as: (RET in the absence of inhibitor-RET
in the presence of inhibitor)/(RET in the absence of inhibitor-RET
in the presence of Leu3a).times.100.
HIV-1 Pseudovirus Assay
[0240] A self-inactivating (SIN) vector was derived from the
pNL4-3.DELTA.Env-luciferase vector (Dragic et al. 1996) by deleting
507 basepairs in the U3 region of the 3' long terminal repeat (LTR)
so as to remove the TATA box and transcription factor binding
sites. The human cytomegalovirus promoter was inserted upstream of
the luciferase (luc) gene to enable expression of luciferase
following integration.
[0241] Reporter viruses pseudotyped with HIV-1.sub.JR-FL or
HIV-1.sub.SF162 envelopes were generated by cotransfection of 293T
cells with the SIN vector and the appropriate pcDNA env-expressing
vector as previously described (Dragic et al. 1996). U87-CD4-CCR5
cells (8,000/well; NIH AIDS Research and Reference Reagent Program)
were infected with 125-375 pg of HIV-1 pseudoviruses in 384-well
plates in the presence or absence of inhibitor(s). Cultures were
incubated for 72 h at 37.degree. C. in DMEM containing 10% fetal
bovine serum, 1 mg/mL puromycin, 0.3 mg/mL geneticin, antibiotics,
and 0.5% DMSO. Luciferase activity (relative light units or RLU)
was measured using BrightGlo reagent (Promega, Madison, Wis.)
according to the manufacturer's instructions. Percent inhibition
was calculated as: (1-RLU in the presence of inhibitor/RLU in the
absence of inhibitor).times.100. IC50 and IC90 were used to denote
the respective concentrations required for 50% and 90% inhibition
of HIV-1.
Synergy Determinations
[0242] Experimental design and data analysis were based on the
combination index (CI) method (Chou et al. 1991; Chou et al. 1984).
Compounds were tested individually and in combination at a fixed
molar ratio over a range of serial dilutions. Entry inhibitors were
combined in equimolar amounts, whereas a 1:10 molar ratio was used
for PRO 140 in combination with azidothymidine and nevirapine.
Dose-response curves were fit using a four-parameter sigmoidal
equation with upper and lower inhibition values constrained to 100%
and 0%, respectively, in order to calculate concentrations required
for 50% (IC.sub.50) and 90% (IC90) inhibition (GraphPad Prism,
GraphPad Software, San Diego, Calif.). CI values for 50% (CI50) and
90% (CI90) inhibition were calculated as previously described (Chou
et al. 1991; Chou et al. 1984). The mutually exclusive CI formula
was used for combinations of CCR5 inhibitors, while the mutually
non-exclusive formula was utilized for combinations of inhibitors
to distinct targets (Chou et al. 1991). Each test was conducted
4-12 times. Synergy, additivity and antagonism are indicated by
CI<1, CI=1 and CI>1, respectively.
Competition Binding Assays
[0243] To examine inhibition of PRO 140 binding, CEM.NKR-CCR5 cells
were suspended in phosphate-buffered saline with 0.1% sodium azide
(PBSA) and incubated with varying concentrations of unlabeled CCR5
antagonists at ambient temperature for 30 minutes. Azide was added
to block CCR5 internalization during the assay. Cells were washed
in PBSA and incubated with 5 nM PRO 140-PE for an additional 30
minutes prior to washing and analysis by flow cytometry using a
FACSCalibur instrument (Becton Dickinson). The extent of PRO 140-PE
binding was measured in terms of both the mean fluorescence
intensity (MFI) and the percent of cells gated for positive
staining.
[0244] To examine inhibition of UK-427,857 binding, CEM.NKR-CCR5
cells were pre-incubated with unlabeled CCR5 inhibitors as
described above prior to addition of 2 nM .sup.3H-UK-427,857 for an
additional 30 minutes. The cells were washed in PBSA and lysed with
0.5N HCl prior to scintillation counting using a Wallac1410
instrument. An additional study reversed the order of addition in
order to examine the stability of UK-427,857 binding over the
course of the assay. Cells were pre-incubated with 2 nM
.sup.3H-UK-427,857 for 30 min prior to washing, addition of
unlabeled inhibitors, and processing as described above. EC50 and
EC90 were used to denote the concentrations of unlabeled compound
required to inhibit binding of labeled compound by 50% and 90%,
respectively.
Statistical Analyses
[0245] Two-tailed t-tests were used to test mean CI50 and CI90
values for the null hypothesis H.sub.0: CI=1 (additivity) using
GraphPad Prism software. P values were corrected for multiple
comparisons from .alpha.=0.05 according to the Bonferroni method
(Cudeck and O'Dell 1994), excluding the PRO 140/PRO 140 mock
combination that was included as an assay control. In the
Bonferroni correction, P=.alpha./n, where n is the number of
comparisons. Twenty-two synergy comparisons (11 compounds.times.2
CI values) were made based on data generated in the membrane fusion
assay, resulting in a corrected P value of 0.0023. In the
pseudovirus assay, 32 synergy comparisons (8 compounds.times.2
viruses.times.2 CI values) resulted in a corrected P value of
0.0016.
Results
Inhibition of HIV-1 Membrane Fusion
[0246] PRO 140 and UK-427,857 were used individually and together
to inhibit HIV-1.sub.JR-FL envelope-mediated membrane fusion in the
RET cell-cell fusion assay, and representative dose-response curves
for the individual agents and combination are illustrated in FIG.
11A. Although both PRO 140 and UK-427,857 individually blocked
HIV-1 fusion at low nanomolar potency, the combination was markedly
more potent. In this assay, 50% inhibition was obtained using 2.9
nM PRO 140 alone, 5.0 nM UK-427,857 used alone, or 2.1 nM of the
combination (1.05 nM PRO 140 plus 1.05 nM UK-427,857). This
supra-additive effect is indicative of antiviral synergy between
the two agents. In contrast, the combination of SCH-D and
UK-427,857 was no more potent than individual agents (FIG. 11B). In
this example, the dose-response curves for the individual
inhibitors and the combination were overlapping, with 50%
inhibition requiring 9.7 nM UK427,857, 5.5 nM SCH-D and 6.1 nM of
the combination. The data suggest purely additive effects for these
inhibitors.
[0247] These studies were extended to additional CCR5 (TAK-779,
RANTES and 2D7), gp120 (BMS-378806 and PRO 542) and gp41
(enfuvirtide) inhibitors, and were repeated four or more times for
each condition. CI50 and CI90 values were calculated for each
condition and averaged across the independent assays. Cooperativity
was assessed using t-tests to determine if the CI50 and CI90 values
were significantly different from one. As a test of these methods,
a PRO 140/PRO 140 mock combination was examined by adding PRO 140
to the assay wells in two separate additions. CI50 and CI90 values
for the PRO 140/PRO 140 combination were 0.96 and 0.97,
respectively (Table 13); therefore, purely additive effects were
observed for this mock combination, as expected.
TABLE-US-00011 TABLE 13 CI values for inhibition of HIV-1.sub.JR-FL
envelope-mediated membrane fusion.sup.a 1.sup.st IC90, Inhibitor
Target IC50, nM nM 2.sup.nd Inhibitor CI50 P value CI90 P value PRO
140 CCR5 2.5 8.6 PRO 140 0.97 .+-. 0.07 0.13 0.96 .+-. 0.14 0.37
UK-427,857 CCR5 5.3 27 PRO 140 SCH-D CCR5 3.2 16 PRO 140 TAK-779
CCR5 11 >200 PRO 140 N/A N/A RANTES CCR5 2.4 38 PRO 140 RANTES
CCR5 2.4 38 UK-427,857 SCH-D CCR5 3.2 16 UK-427,857 0.86 .+-. 0.03
0.016 0.75 .+-. 0.02 0.0033 SCH-D CCR5 3.2 16 TAK-779 1.3 .+-. 0.18
0.12 N/A N/A 2D7 CCR5 3.7 58 PRO 140 1.0 .+-. 0.14 0.61 1.9 .+-.
0.61 0.024 enfuvirtide gp41 8.6 66 PRO 140 0.84 .+-. 0.16 0.040
0.89 .+-. 0.20 0.19 PRO 542 gp120 8.9 91 PRO 140 0.96 .+-. 0.17
0.56 0.94 .+-. 0.19 0.45 BMS-378806 gp120 5.2 20 PRO 140 1.1 .+-.
0.22 0.19 .sup.aStatistically significant results (P < 0.0023
after application of the Bonferroni correction for multiple
comparisons) are indicated in italicized bold text. IC50 and IC90
denote values for the 1.sup.st inhibitor. N/A = not applicable;
TAK-779 did not consistently achieve 90% inhibition in the assay.
CI values represent the means and standard deviations of 4-12
independent assay
[0248] Potent synergy was observed for PRO 140 in combination with
each of three small-molecule CCR5 antagonists (UK427,857, SCH-D and
TAK-779), and the findings were statistically significant even when
the data were corrected for multiple comparisons via the Bonferroni
method (Table 13). CI values ranged from 0.36 to 0.61, and these
synergies translated into dose reductions ranging from 3- to 8-fold
across the different conditions. Synergies were greater at 90%
inhibition than at 50% inhibition. Synergy between PRO 140 and
small-molecule CCR5 antagonists was robust in that it was observed
at both the 50% and 90% inhibition levels in every instance. The
exception was TAK-779, which did not mediate 90% inhibition when
used individually, and therefore a CI90 was not determined.
Similarly potent synergy was observed when RANTES was used in
combination with either PRO 140 or UK-427,857 (Table 13).
Additional tests examined combinations of two small-molecule CCR5
antagonists (SCH-D/UK-427,857 and SCH-D/TAK-779) or two CCR5 mAbs
(PRO 140/2D7). No significant synergy was observed for these
combinations, although the SCH-D/UK-427,857 CI90 values trended
towards significance. The findings are consistent with prior
observations of overlapping binding sites for PRO 140 and 2D7
(Olson et al. 1999) and for SCH-D and TAK-779 (Seibert et al.
2006). PRO 140 was also tested in combination with the gp41 fusion
inhibitor enfuvirtide and with the gp120 attachment inhibitors PRO
542 and BMS-378806 (Table 13). CI values ranged from 0.84 to 1.28,
and none of these combinations demonstrated synergy that met the
criteria for statistical significance. For the PRO 140/BMS-378806
combination, modest antagonism was observed at 50% but not 90%
inhibition. The biological significance of this result is
unclear.
Inhibition of HIV-1 Pseudoviruses
[0249] Single-cycle HIV-1 reporter viruses were used to examine
whether the synergistic effects were limited to cell-cell fusion or
whether they extended to other modes of HIV-1 entry. Signals in
this assay require both viral entry and reverse transcription, so
that both NRTI and NNRTI may be included in the analyses. Each
combination was tested against reporter viruses pseudotyped with
envelopes from HIV-1.sub.JR-FL and HIV-1.sub.SF162 in at least 4
independent assays per virus. A PRO 140/PRO 140 mock combination
was again included as an assay control, and demonstrated additive
effects against both HIV-1.sub.JR-FL and HIV-1.sub.SF162
pseudoviruses, as expected (Table 14).
[0250] PRO 140 potently synergized with both UK-427,857 and SCH-D
in blocking virus-cell fusion, and the results met the criteria for
statistical significance. Comparable levels of synergy were
observed against both HIV-1.sub.JR-FL and HIV-1.sub.SF162
pseudoviruses at 50% and 90% inhibition (Table 14), with CI values
ranging from 0.18 to 0.64. These synergies translated into dose
reductions ranging to 14-fold. These results are in good agreement
with those obtained in the cell-cell fusion assay (Table 13).
Neither TAK-779 nor RANTES mediated consistent, high-level
inhibition of HIV-1 pseudovirus entry, and therefore these
compounds were not included in this analysis (data not shown).
TABLE-US-00012 TABLE 14 CI values for inhibition of HIV-1 reporter
viruses pseudotyped with envelopes from HIV-1.sub.JR-FL and
HIV-1.sub.SF162.sup.a. HIV-1 IC50, IC90, 1.sup.st Inhibitor Target
Envelope nM nM 2.sup.nd Inhibitor CI50 P value CI90 P value PRO 140
CCR5 JRFL 2.2 28 PRO 140 1.2 .+-. 0.32 0.16 0.90 .+-. 0.15 0.047
SF162 1.3 20 PRO 140 1.0 .+-. 0.27 1.0 0.86 .+-. 0.33 0.21 SCH-D
CCR5 JRFL 2.4 44 PRO 140 SF162 0.34 14 PRO 140 UK-427,857 CCR5 JRFL
7.4 46 PRO 140 SF162 0.87 13 PRO 140 UK-427,857 CCR5 JRFL 7.4 46
SCH-D 0.71 .+-. 0.11 0.16 1.2 .+-. 0.15 0.32 SF162 0.87 13 SCH-D
0.87 .+-. 0.06 0.19 0.86 .+-. 0.28 0.61 2D7 CCR5 JRFL 8.8 >200
PRO 140 1.5 .+-. 0.25 0.024 N/A N/A SF162 2.2 74 PRO 140 1.1 .+-.
0.47 0.61 1.0 .+-. 0.16 0.65 PRO 542 gp120 JRFL 0.19 2.9 PRO 140
1.2 .+-. 0.32 0.22 1.0 .+-. 0.18 0.92 SF162 0.36 7.1 PRO 140 0.98
.+-. 0.28 0.84 0.64 .+-. 0.26 0.010 BMS-378806 gp120 JRFL 1.2 11
PRO 140 1.2 .+-. 0.38 0.43 0.74 .+-. 0.23 0.059 SF162 0.03 0.42 PRO
140 1.1 .+-. 0.28 0.36 0.82 .+-. 0.21 0.068 nevirapine RT JRFL 30
310 PRO 140 1.2 .+-. 0.38 0.36 0.73 .+-. 0.28 0.068 SF162 42 280
PRO 140 1.2 .+-. 0.34 0.30 0.63 .+-. 0.19 0.033 zidovudine RT JRFL
140 1900 PRO 140 1.1 .+-. 0.38 0.37 0.85 .+-. 0.26 0.21 SF162 86
2100 PRO 140 0.99 .+-. 0.27 0.91 1.0 .+-. 0.38 1.0
.sup.aStatistically significant results (P < 0.0016 after
application of the Bonferroni correction for multiple comparisons)
are indicated in italicized bold text. IC50 and IC90 refer to
values for the 1.sup.st inhibitor. N/A = not applicable; 2D7 did
not consistently achieve 90% inhibition in the assay. CI values
represent the means and standard deviations of 4 or more
independent assays
[0251] Additive effects were observed for both the UK-427,857/SCH-D
and PRO 140/2D7 combinations (Table 14). Similarly, additivity was
observed for PRO 140 used in combination with the gp120 inhibitors
PRO 542 and BMS-378806. No antagonism was observed for the PRO
140/BMS-378806 combination against either virus. Overall, these
findings are consistent with those seen for cell-cell fusion.
Lastly, additive effects were observed for PRO 140 in combination
with either zidovudine (NRTI) or nevirapine (NNRTI).
Competition Binding Studies
[0252] As described above, additive antiviral effects were observed
for inhibitors known (PRO 140 and 2D7) or inferred (UK-427,857 and
SCH-D) to compete for CCR5 binding; however, little is known
regarding the competitive binding of synergistic compounds (e.g.,
PRO 140/UK-427,857 and PRO 140/SCH-D). Since non-competitive
binding provides a possible mechanism for synergy between CCR5
inhibitors, this issue was explored using labeled forms of
UK-427,857 and PRO 140.
[0253] Flow cytometry was used to examine inhibition of PRO 140-PE
binding to CEM.NRK.CCR5 cells by unlabeled PRO 140, UK-427,857 and
SCH-D. PRO 140-PE binding was efficiently inhibited by unlabeled
PRO 140, as expected. Complete inhibition was observed in terms of
both MFI values (FIG. 12A) and the percent of cells gated for
positive binding (FIG. 12B). The EC50 based on MFI data was 2.5 nM
(FIG. 12A), and this value compares favorably with the antiviral
IC50 of PRO 140 (Tables 13 and 14). Since percent cells gated is a
readout for essentially complete inhibition of binding, an EC90
value was calculated as 17 nM, and this value is similar to the
antiviral IC90 values observed for PRO 140 (Tables 13 and 14). 2D7
also completely inhibited binding of PRO 140-PE to CEM.NKR-CCR5.
The CCR5 specificity of PRO 140-PE was also demonstrated by its
inability to bind parental CEM.NKR cells.
[0254] In sharp contrast, modest levels of inhibition were observed
for UK427,857 and SCH-D (FIG. 12). Micromolar concentrations of
UK-427,857 and SCH-D reduced PRO 140-PE MFI values by 50% or less
(FIG. 12A). More dramatically, UK-427,857 and SCH-D had little
impact on the percent of cells gated for positive binding of PRO
140-PE (FIG. 12B). The findings suggest that UK427,857 and SCH-D
partially reduce the number of PRO 140-PE molecules bound per cell;
however, these compounds do not reduce the number of cells that
bind measurable amounts of PRO 140-PE. Therefore, UK427,857 and
SCH-D represent partial antagonists of PRO 140 binding, and this
finding provides a mechanism for the antiviral synergy observed
between PRO 140 and these small-molecule CCR5 antagonists.
[0255] Inhibition of .sup.3H-UK427,857 binding by unlabeled
UK427,857, SCH-D and PRO 140 was next examined. Binding of
.sup.3H-UK427,857 to CEM.NKR-CCR5 cells was efficiently inhibited
by unlabeled UK427,857 (FIG. 13A). The EC50 for binding was 4.3 nM
and is similar to the antiviral IC50 values observed for UK-427,857
(Tables 13 and 14).
[0256] SCH-D also blocked .sup.3H-UK427,857 binding to background
levels (FIG. 13A). However, there was no correlation between the
compounds' antiviral potency and their potency in blocking
.sup.3H-UK427,857 binding. For example, whereas SCH-D demonstrated
equal or slightly greater antiviral potency than UK427,857 (Tables
13 and 14), SCH-D was less potent in blocking .sup.3H-UK-427,857
binding (EC50=17 nM, FIG. 13A). This result is consistent with
minor differences in the CCR5 binding sites of these compounds.
[0257] Surprisingly, PRO 140 also blocked .sup.3H-UK427,857 binding
to background levels (FIG. 13A), and this result contrasts with the
modest inhibition of PRO 140-PE binding by UK-427,857 (FIG. 12).
PRO 140 inhibited .sup.3H-UK-427,857 binding with an EC50 of 14 nM,
which is 5-10 fold higher than the antiviral IC50 of PRO 140
(Tables 13 and 14).
[0258] A final experiment examined the stability of UK-427,857
binding to CEM.NKR-CCR5 cells under the conditions of the
competition assay. For this, cells were pre-incubated with
.sup.3H-UK-427,857 and then the dissociation was examined in the
presence of unlabeled UK427,857, SCH-D and PRO 140. As indicated in
FIG. 13B, there was minimal dissociation of .sup.3H-UK-427,857 over
30 min at ambient temperature, and UK427,857 wasn't displaced by
either PRO 140 or SCH-D. Therefore, the inability of UK-427,857 to
efficiently compete PRO 140 binding to CCR5 (FIG. 12) is not due to
rapid dissociation of UK-427,857 from CCR5 during the course of the
assay. Collectively, the data indicate that PRO 140 can bind CCR5
in the presence of pre-bound UK-427,857.
Discussion
[0259] This study explores interactions between mAb and
small-molecule CCR5 inhibitors and examines combinations of CCR5
drugs that currently are in development for HIV-1 therapy.
Surprisingly, potent antiviral synergy between the CCR5 mAb PRO 140
and each of three structurally distinct small-molecule CCR5
antagonists was observed. Consistent, high-level synergy was
observed across varying assay systems, viral isolates, target cells
and inhibition levels. PRO 140 and small-molecule CCR5 antagonists
were more potently synergistic when used together rather than in
combination with inhibitors that block other stages of HIV-1 entry.
In contrast, additive effects were observed for combinations of two
small-molecule CCR5 antagonists. Competition binding studies
revealed complex and non-reciprocal patterns of CCR5 binding by mAb
and small-molecule CCR5 inhibitors, and suggest that the
synergistic interactions occur at the level of receptor
binding.
[0260] Robust synergy between mAb and small-molecule CCR5
inhibitors was observed in this study. Potent synergy was observed
for both cell-cell and virus-cell fusion, and there was a good
concordance of findings in these two well-established assay
systems. Comparable levels of synergy were observed for PRO 140 in
combination with each of 3 small-molecule CCR5 antagonists from
unrelated chemical series. In addition, consistent synergy was
observed for each of two well-characterized HIV-1 envelopes and two
CCR5 target cells. Synergy increased with increasing levels of
viral inhibition and translated into in vitro dose reductions of up
to 14-fold. Viewed alternatively, this degree of synergy provides a
corresponding increase in antiviral pressure at a given
concentration of drugs, thereby improving viral suppression and
potentially delaying the emergence of drug-resistant virus. This is
supported by preliminary studies indicating the mAb and
small-molecule CCR5 inhibitors possess complementary patterns of
viral resistance (Kuhmann et al. 2004 and Marozsan et al. 2005).
The present findings provide a rationale for clinical exploration
of regimens that combine mAb and small-molecule CCR5
inhibitors.
[0261] Potent synergy was also observed for RANTES used in
combination with either UK-427,857 or PRO 140. Endogenous levels of
RANTES may afford some protection against HIV-1 disease progression
during natural infection (Garzino-Demo et al. 1999; Lui et al.
1999), and therefore this finding of synergy has important and
positive implications for CCR5-targeted therapies of HIV-1.
Antiviral synergy between RANTES and PRO 140 is not surprising
based on a prior observation that RANTES signaling is not blocked
by antiviral concentrations of murine PRO 140 (PA14) (Olson et al.
1999).
[0262] Synergy between RANTES and UK-427,857 is less easily
explained given that UK427,857 is a potent CCR5 antagonist.
However, these findings are consistent with prior observations of
synergy between the small-molecule CCR5 antagonist SCH-C and
aminooxypentane-RANTES (AOP-RANTES) (Tremblay et al. 2002), a
RANTES derivative that has been evaluated as a potential topical
microbicide (Kawamura et al. 2000).
[0263] In contrast to the robust synergy observed between mAb and
small-molecule CCR5 antagonists, additive effects were observed for
combinations of small-molecule CCR5 antagonists. Lack of
cooperativity is consistent with the view that these molecules
compete for binding to a common pocket on CCR5 (Dragic et al. 2000;
Nishikawa et al. 2005; Tsamis et al. 2003; Watson et al. 2005). The
in vitro studies do not provide a basis for combining
small-molecule CCR5 antagonists in the clinic based solely on
inhibition of wild-type virus.
[0264] Similarly, potent synergy was not observed between PRO 140
and inhibitors of HIV-1 attachment (PRO 542 and BMS-378806), fusion
(enfuvirtide), or reverse transcriptase (zidovudine and
nevirapine), and these findings underscore the significance of the
synergy observed for PRO 140 and small-molecule CCR5 antagonists. A
number of prior studies have examined interactions between various
small-molecule CCR5 antagonists (UK-427,857, SCH-C, TAK-220,
TAK-652 and E913) and drugs from each of the existing HIV-1
treatment classes. Most (Tremblay et al. 2005 Antivir. Ther.;
Tremblay et al. 2005 Antimicrob. Agents Chemother; Tremblay et al.
2002) but not all (Dorr et al. 2005; Maeda et al. 2001) studies
have reported broad synergy between CCR5 inhibitors and the other
HIV-1 treatment classes, and the divergent results may reflect
differences in the compounds and methods used for antiviral testing
as well as differences in the methods used for data analysis. When
UK427,857 was tested against 20 licensed antiretroviral agents,
additive effects were observed in all but three cases, where modest
synergy was reported (Dorr et al. 2005). This result is consistent
with the present findings for combinations of PRO 140 and HIV-1
inhibitors that do not target CCR5.
[0265] Without intending to be bound by theory, synergy between
anti-HIV-1 drugs may stem from a variety of mechanisms. In mixed
virus cultures, one compound may inhibit virus resistant to a
second compound (Johnson et al. 1991), and NRTI NNRTI combinations
may overcome specific RT-mediated resistance mechanisms
(Basavapathruni et al. 2004; Borkow et al. 1999). Metabolic
interactions between inhibitors may increase their effective
intracellular drug concentrations (Molla et al. 2002), and
synergistic entry inhibitors may disrupt interdependent steps in
the entry cascade (Nagashima et al. 2001; Tremblay et al. 2000).
The present study examined clonal viral envelopes rather than mixed
populations, and the extracellular nature of the target argues
against metabolic interactions. Multiple domains of gp120
contribute to CCR5 binding (Cormier et al. 2002), but it is unclear
at present whether these interactions represent separate or
discrete events during infection.
[0266] The present findings indicate that antiviral synergy between
mAb and small-molecule CCR5 inhibitors may occur at the level of
the receptor. As discussed above, mAbs and small molecules bind
distinct loci on CCR5 (Dragic et al. 2000; Nishikawa et al. 2005;
Tsamis et al. 2003; Olson et al. 1999; Watson et al. 2005). When
pre-incubated with CCR5 cells in the present study, PRO 140
completely blocked subsequent binding of UK-427,857 to the
receptor; although the PRO 140 concentrations were higher than
those needed to block HIV-1 entry into the same cells. In contrast,
pre-incubation of CCR5 cells with super-saturating concentrations
of UK-427,857 or SCH-D reduced PRO 140 binding by 50% or less. As
one possible explanation, PRO 140 could recognize CCR5 conformers
that are not bound by UK-427,857 or SCH-D. Although cell-surface
CCR5 exists in multiple conformations (Lee et al. 1999), it seems
unlikely that the small-molecule antagonists could demonstrate
potent antiviral activity while failing to bind a significant
fraction of cell-surface CCR5. In this regard, it is important to
note that a common cellular background (CEM.NKR-CCR5 cells) was
used for competition binding and antiviral studies, and therefore
the findings are not related to cell-specific differences in CCR5
expression.
[0267] Without intending to be bound by theory, another plausible
explanation for the present findings is that PRO 140 is capable of
forming a ternary complex with UK-427,857-bound CCR5, and this
ternary complex provides an increased barrier to HIV-1 entry.
Within the context of this model, PRO 140 may bind UK427,857-bound
CCR5 somewhat less efficiently than free CCR5, as evidenced by the
modest reduction in PRO 140 binding in the presence of
UK-427,857.
[0268] The combination index method is widely used to assess
drug-drug interactions. In this method, cooperativity often is
defined on the basis of empirical CI values (e.g., <0.9 for
synergy and >1.1 for antagonism) irrespective of inter-assay
variability. Statistical analyses are performed infrequently, and
even more rarely are adjustments made for multiple comparisons. In
the absence of such analyses, there is increased potential to
overestimate the number of synergistic combinations.
[0269] A rigorous and conservative approach to identifying
synergistic effects was employed. CI values were tested for
statistical significance against the null hypothesis of additivity
(CI=1). In addition, these studies determined 20-30 different CI
values per experiment (Tables 13 and 14), as is common in synergy
studies. In order to reduce the potential for spurious positive
results, the significance level was reduced using the Bonferroni
correction. A mock combination was also evaluated as a test of
these methods for antiviral testing and data analysis. It was
therefore concluded that numerous apparent synergies (CI<0.9)
could not be distinguished from inter-assay variation based on the
available data. However, despite the rigorous nature of these
methods, PRO 140 and small-molecule inhibitors demonstrated
significant synergy under every test condition, lending credence to
this finding. Combinations with CI values that trended towards
significance in the present survey could be explored in future
studies. For example, data for the PRO 140/enfuvirtide combination
suggested modest synergy that trended towards significance; thus
this combination may also be useful for treating HIV-1
infection.
[0270] A growing body of data indicates that mAb and small-molecule
CCR5 antagonists represent distinct subclasses of CCR5 inhibitors,
and a number of important parallels can be drawn between NRTI and
NNRTI on the one hand and between mAb and small-molecule CCR5
antagonists on the other. In each instance, there are distinct
binding loci for the inhibitors on the target protein (reverse
transcriptase or CCR5). One set of inhibitors (NNRTI or
small-molecule CCR5 antagonists) acts via allosteric mechanisms,
while the other set (NRTI or CCR5 mAbs) acts as a competitive
inhibitor. Like NRTI and NNRTI, mAb and small-molecule CCR5
inhibitors are synergistic and possess complementary patterns of
viral resistance in vitro in preliminary testing (Kuhmann et al.
2004; Marozsan et al. 2005). NRTI and NNRTI represent important and
distinct treatment classes even though they target the same
protein, and mAb and small-molecule CCR5 inhibitors similarly may
offer distinct HIV-1 treatment modalities.
Part IV
Materials and Methods
[0271] PRO 140 and small-molecule CCR5 antagonists were prepared
and/or obtained as described herein above. The primary R5 HIV-1
isolates JR-FL and Case C 1/85 (CC1/85) were passaged weekly in
vitro on peripheral blood mononuclear cells (PBMCC) in the presence
or absence of progressively increasing concentrations of PRO 140 or
SCH-D, and viral cultures were examined for susceptibility to these
and other CCR5 inhibitors. For susceptibility testing, viruses were
cultured in vitro on stimulated PBMC. In the presence and absence
of serially diluted drug, and the extent of viral replication was
determined by p24 ELISA.
Results
[0272] For both JR-FL and CC1/85, drug-resistant variants were
generated in the presence of PRO 140 and SCH-D. At passage 12, the
escape mutants were approximately 10- to 100-fold less susceptible
to the drug used for selection. In each case, the escape mutants
continued to require CCR5 for replication on PBMC. Complementary
patterns of resistance were observed: SCH-D escape mutants were
efficiently inhibited by PRO 140 and PRO 140 escape mutants were
efficiently inhibited by SCH-D.
Discussion
[0273] PRO 140 escape mutants continue to require CCR5 for entry
and remain susceptible to small-molecule CCR5 antagonists. In
addition, PRO 140 is active against viruses resistant to
small-molecule CCR5 antagonists. These findings indicate that PRO
140 and small-molecule CCR5 antagonists may represent distinct
subclasses of CCR5 inhibitors.
Part V
Phase 1b Clinical Trial:
[0274] A Phase 1b, double-blind, randomized, single-dose,
dose-cohort escalation study was conducted in which PRO 140 or
placebo control was administered intravenously to adult (male and
female) HIV-infected subjects. The efficacy data collected during
the study were changes in viral load and CD4 counts over time. The
safety data collected during the study were serious adverse events
(SAEs)/adverse events (AEs) and changes in laboratory parameters
(hematology, chemistry), physical exam, viral tropism and ECGs over
time. The exploratory data collected included PK, immunogenicity
(anti-PRO 140 antibody production), RANTES, and CCR5 lymphocyte
coating over time.
Clinical Trial Design and Study Results
[0275] This multi-center, double-blind, randomized,
placebo-controlled phase 1b trial examined three single intravenous
escalating doses of PRO 140: 0.5 mg/kg, 2.0 mg/kg and 5.0 mg/kg.
The study was designed to assess the safety, tolerability,
pharmacology and antiviral activity of PRO 140 through day 59 and
was conducted at 10 sites in the United States. Thirty-nine
HIV-infected individuals who had taken no anti-retroviral therapy
within the preceding three months and who had plasma HIV RNA levels
(viral loads) greater than or equal to 5,000 copies/mL were
enrolled to receive PRO 140 monotherapy or placebo. The
HIV-infected individuals in the study had a CD4+ count of >250
cells/.mu.g. All patients were screened prior to the study for the
presence of virus that utilizes only CCR5 as the entry coreceptor.,
i.e., CCR5-tropic virus. Of the 13 patients in each cohort, 10
patients received PRO 140 and three received placebo (10:3). A
summary of the baseline characteristics of the patients in the
study is presented in Table 15.
TABLE-US-00013 TABLE 15 Subject Baseline Characteristics Placebo
0.5 mg/kg 2 mg/kg 5 mg/kg All Patients Characteristic (n = 9) (n =
10) (n = 10) (n = 10) (n = 39) Age 40.3 37.1 37.6 42.8 40.3 median
(range) (23.8-50.2) (24.1-53.2) (23.2-51.5) (22.9-61.1) (22.9-61.1)
Gender (n) 8/1 10/0 8/2 5/5 31/8 male/female Race (n) 4/5/0 4/4/2
4/6/0 5/4/1 17/19/3 black/white/other Weight, kg 81.4 81.0 81.7
73.4 80.9 median (range) (57.3-101.7) (54.2-111.4) (55.9-142.9)
(52.7-86.8) (52.7-142.9) CD4, cells/.mu.l) 439 492 438 535 484
median (range) (281-555) (443-762) (269-613) (303-853) (269-853)
Log.sub.10 HIV RNA, 4.44 4.45 4.44 4.37 4.43 copies/mL (3.98-5.61)
(3.79-5.54) (3.89-4.94) (3.81-5.36) (3.79-5.61) median (range)
[0276] The primary efficacy endpoint was the reduction in plasma
HIV RNA level as measured by the Roche Amplicor.TM. Assay. The
primary efficacy endpoint is the maximum change from baseline in
viral load, defined as HIV-1 copies/ml, as measured by the Roche
Amplicor.TM. Assay. In the Phase 1b study, the results were
positive, dose-dependent, and highly statistically significant for
the two highest doses tested. HIV-infected individuals who received
5.0 mg/kg of PRO 140 achieved an average maximum decrease of viral
load of 1.83 log.sub.10 (98.5%; P<0.0001), with individual
reductions ranging up to 2.5 log.sub.10 (99.7%) at the 2.0 mg/kg
and 5.0 mg/kg dose levels. At nine days post-treatment, mean HIV
RNA values nadired, and these same individuals achieved a mean
viral load reduction of 1.70 log.sub.10 (98%; P<0.0001). At this
time, mean PRO 140 serum concentrations were 1.4 and 4.1 .mu.g/ml
in the 2.0 mg/kg and 5.0 mg/kg dose levels, respectively. In the
5.0 mg/kg cohort, mean viral load was suppressed by 1.0 log.sub.10
(90%) within four days of dosing and persisted at or below the 1.0
log.sub.10 level of reduction for two to three weeks in patients
before returning to baseline at approximately 30 days. The response
rate among the treatment groups (percentage of patients with a
.gtoreq.1 log.sub.10 decrease in HIV RNA at any time) increased
with PRO 140 dose, reaching a maximum of 100% in the highest dose
cohort (P<0.0001).
[0277] Mean log.sub.10 HIV RNA changes of -0.13, -0.37, -1.04
(P=0.0001) and -1.70 (P=0.0001) were observed at Day 10
post-treatment for the placebo, 0.5 mg/kg, 2.0 mg/kg and 5.0 mg/kg
groups, respectively, as shown in FIG. 22; and a >10-fold
decrease in HIV RNA was observed in 0/9, 1/10, 6/10 (P=0.01) and
10/10 (P=0.0001) individuals in these respective treatment groups.
All PRO 140-treated individuals had exclusively R5 virus at
pre-dose (baseline) and at end of study; there was no change in
viral susceptibility to PRO 140 during the course of the study.
Antiviral effects were evaluated as functions of PRO 140 serum
levels, CCR5 receptor occupancy and viral susceptibility.
[0278] The conclusions determined from this Phase 1b study are
presented below: [0279] PRO 140 at single doses of 2.0 mg/kg and
5.0 mg/kg were effective in reducing viral load. [0280] A dose
response and an ineffective dose were identified. [0281] PRO 140 5
mg/kg efficacy was dramatic, as evidenced by: [0282] Maximum
decrease of viral load at any time is -1.8 log, p<0.0001, as
shown in FIGS. 19 and 21. [0283] Viral load decrease was
statistically significant by day 5, remained statistically
significant through day 15 (day 22 was -0.73, p=0.052), nominal p
values, as shown in FIG. 19. [0284] Patients with >1 log
decrease (anytime) 10/10 (100%), p<0.0001, as shown in FIG. 18.
[0285] AUC viral load is significantly decreased (p=0.022), as
shown in FIG. 18. [0286] Patients with <400 copies/mL (anytime)
4/10 (40%), p=0.087, as shown in FIG. 18. [0287] At 5 mg/kg,
transient rise in and trend toward increased CD4+ lymphocytes (129
cells/mm.sup.3 (29%) average increase at Day 8), p=0.055, nominal p
values as shown as shown in FIGS. 20A and 20B. Levels remained
elevated for 3 weeks post-treatment. [0288] Safety [0289] PRO 140
doses were well-tolerated in all study groups. [0290] No treatment
related SAEs and no apparent dose-related AEs were observed. [0291]
No dose-limiting toxicity or obvious pattern of toxicity was
observed. [0292] No change in plasma RANTES (CCL5) chemokine levels
was observed. [0293] No difference relative to placebo in HIV
co-receptor tropism. No tropism shift (e.g., from CCR5 tropism to
CXCR4 tropism) on treatment with PRO 140. All subjects screened for
R5-only virus. Dual/mixed tropism result observed post-treatment in
1/9 placebo subjects (11%) and in 1/30 PRO 140 subjects (3%; 0.5
mg/kg group). [0294] Immunogenicity [0295] One patient in the 5
mg/kg cohort had a positive titer (1:40) for anti-PRO 140 antibody
at day 59. [0296] All other subjects in all cohorts tested
negative. [0297] Pharmacokinetics (PK) [0298] Serum concentrations
of PRO 140 and anti-PRO 140 antibodies measured by ELISA. (FIG.
26). [0299] Concentrations [of PRO 140 in plasma] are 1% of Cmax by
days 6-7. [0300] PK metrics assessed by non-compartmental analysis.
Peak and total exposure increased proportionally or better with
dose. Terminal half life approximately 4 d. [0301] Low titer
anti-PRO 140 antibodies in one subject (in 5 mg/kg group). No
obvious effect on PK or antiviral response. [0302] Coating of CCR5
Lymphocytes [0303] Lymphocytes analyzed by flow cytometry ex vivo
with fluorescently-labeled PRO 140 and non-competing CCR5 antibody.
[0304] No depletion of CCR5+ lymphocytes. [0305] Obvious coating of
CCR5 lymphocytes by PRO 140. [0306] Duration of coating maximal for
1-2 weeks and consistent with duration of antiviral effects. (FIG.
27). [0307] Virus Susceptibility [0308] All viruses in PRO
140-treated subjects were susceptible to PRO 140 at baseline, with
minimal variation. Mean rIC50=2.0 (range: 0.83-5.1). Mean Maximum
Percent Inhibition (MPI)=99% (range: 93-100%). [0309] No change in
susceptibility post-treatment. Less than 2-fold change in rIC50 for
all subjects. Mean rIC50=2.1 (range: 0.98-4.71). Mean MPI=99%
(range: 93-100%). (FIG. 25). [0310] No change from baseline to day
59 in either the IC50 or the Fold Change of PRO 140 or Fuzeon, as
determined by the PhenoSense.TM. Assay. [0311] Correlates of
Efficacy [0312] PRO 140 dose was correlated with both the magnitude
of HIV-1 RNA reduction (p=0.0001) and the duration of the response
(p=0.0059). [0313] Correlation between HIV-1 RNA reduction and PRO
140 exposure (E.sub.max analysis). (FIG. 28). [0314] HIV-1 RNA
nadirs were independent of baseline HIV-1 RNA, baseline CD4+ cells
and baseline CCR5+ cells. Accordingly, antiviral effects correlated
with PRO 140 dose and exposure, but not with baseline HIV-1 RNA,
CD4 cells, or CCR5 cells. (FIG. 29) [0315] Preclinical studies
support clinical use and feasibility of SC delivery [0316] Safety
and tolerability of SC administration in a six-month, preclinical
animal model study. [0317] Repeat SC dosing feasible. [0318] Weekly
and q2weeks SC dosing expected to provide exposure similar to that
of q2 weeks 5 mg/kg IV dose and may reduce differences in peak and
trough concentrations.
[0319] The virological response rate was determined at the
completion of the Phase 1b study, as shown in FIG. 23. Coreceptor
tropism results (Trofile.TM., Monogram Biosciences), are shown in
FIG. 24. For the placebo cohort, 1/9 subjects (11%, days 1, 8, 29
and 59) showed dual/mixed (D/M) tropism results. For the 0.5 mg/ml
PRO 140 cohort, 1/30 subjects (3%, day 8 only) showed D/M tropism.
Analysis of Env clones of pre-dose and D/M viruses can determine
whether such low frequency of D/M tropism results from clones
(CXCR4) that pre-existed PRO 140 treatment and which are
phylogenetically highly related to on-treatment CXCR4-using virus.
Where no CXCR4-using clones are identified in a pre-dose sample
from a subject, the on-treatment CXCR4-using virus can be
identified as being phylogenetically very distant from the CCR5
tropic baseline virus; in such a case, the emergence of a pre-dose
or pre-treatment CXCR4-using virus, i.e., one that is present at
baseline, but is not detected, may likely explain the D/M tropism
in the subject, as opposed to a tropism switch.
Part IV
[0320] PRO 140 and PA14, the murine precursor to PRO 140, were
tested for the ability to inhibit replication of five CCR5-tropic
(R5) HIV-2 isolates and two mixed/dual-tropic (R5X4) HIV-2 isolates
in human peripheral blood mononuclear cells (PBMC). Two
well-characterized R5 HIV-1 isolates were included for comparative
purposes.
Materials and Methods:
[0321] Viruses: All virus isolates were grown and titered
exclusively in stimulated PBMC as described previously.
HIV-1.sub.JR-FL and HIV-1.sub.cc 1/85, formerly known as
HIV-1.sub.Case C 1/85 have been studied previously for
susceptibility to PRO 140 and PA14. The HIV-2 isolates used in this
study have been previously described (Owen, S. M., Ellenberger, D.,
Rayfield, M., Wiktor, S., Michel, P., Grieco, M. H., Gao, F., Hahn,
B. H., and Lai, R. B. 1998. Genetically Divergent Strains of Human
Immunodeficiency Virus Type 2 Use Multiple Coreceptors for Entry. J
Virol 72:5425-5432) and were obtained from the NIH AIDS Research
and Reference Reagent Program.
[0322] Inhibitors: PRO 140 (Lot #4027-COO) was produced in
accordance with Good Manufacturing Practice. PA14 (Lot # DF-021204)
was produced for reagent use in the Research & Development
laboratories of Progenics Pharmaceuticals Inc.
[0323] Inhibition of HIV-1 and HIV-2 replication in PBMC cultures:
PBMC were isolated from 34 healthy blood donors with Ficoll-Hypaque
(all media from Cellgro, Herndon, Va.) and depleted of CD8+ cells
using RosetteSep CD8 depletion cocktail (StemCell Technologies)
according to the manufacturer's instructions. Equal numbers of
cells from each donor were then separately stimulated with 5 ug
phytohemagglutinin/ml (Sigma, St. Louis, Mo.) or with
surface-immobilized anti-CD3 antibody OKT3 in RPMI 1640 medium
containing 10% fetal calf serum, 100 Units of interleukin-2/ml (NIH
AIDS Research and Reference Reagent Program), glutamine, and
antibiotics (PBMC culture medium) as previously described. After 72
hours, equal numbers of PBMC stimulated by one of these two methods
from each donor were combined for use in infection assays as
follows. PBMC (1.4.times.10.sup.5) in 100 ul of PBMC culture medium
were combined with 50 ul aliquots of serially diluted inhibitor for
1 h at 37.degree. C. The virus inoculum was adjusted to 400 to
2,000 times the TCID.sub.50 per ml in PBMC culture medium, and a
50-ul aliquot was added to each culture. The inhibitory doses refer
to the concentrations of the agents present at this point in the
culture, when virus, cells, and inhibitors were all present at
their final concentrations. Reverse Transcriptase (RT) activity was
measured on Day 7 post infection (pi) for HIV-1 and Day 10 pi for
HIV-2 by the Reverse Transcriptase Assay, Colorimetric kit, #11 468
120 910, Roche (Switzerland) according to the manufacturer's
instructions with the following exception: neither
ultracentrifugation nor PEG precipitation of the culture
supernatants was performed before viral lysis.
[0324] The inhibition data were analyzed using GraphPad Prism
software (GraphPad Software, San Diego, Calif.). A 4-parameter
logistic fit was used calculate the concentration of inhibitor that
afforded a 50% and a 90% reduction in RT activity. These were
designated the 50% and 90% inhibitory concentrations (EC.sub.50 and
EC.sub.90). The % inhibitions where best-fit curve reached a top
plateau, also as calculated by GraphPad Prism software, are
reported as well as maximum percent inhibition (MPI). Inhibition
assays were repeated 5 times, and these data were used to determine
median EC.sub.50, EC.sub.90 and MPI values. Inhibition curves with
a poor sigmoid fit (R.sup.2<0.7) were excluded from data
analysis. In assays where <50% or <90% inhibition was
obtained at the highest concentration of inhibitor tested (or
>50% or >90% were obtained at the lowest concentration of
inhibitor tested), the highest (or lowest) test concentration from
that assay was used to calculate the median EC.sub.50 or
EC.sub.90.
Results:
[0325] PRO 140 and PA14 were tested against five R5 HIV-2 isolates.
For comparison, PRO 140 and PA14 were also tested against two
mixed/dual-tropic (R5X4) HIV-2 isolates, and two R5 HIV-1 reference
isolates. The median concentrations of PRO 140 and PA14 that
afforded 50% and 90% reductions in viral growth (EC.sub.50 and
EC.sub.90, respectively) are listed below in Table 16. PRO 140
inhibited replication of each of the five R5 HIV-2 isolates in this
study, and the susceptibility of HIV-2 was similar to that of the
reference R5 HIV-1 isolates. PRO 140 and PA14 showed comparable
activities in this study. Neither PRO 140 nor PA14 measurably
affected replication of R5X4 HIV-2 isolates.
TABLE-US-00014 TABLE 16 Median PRO 140 and PA14 EC.sub.50 and
EC.sub.90 values for HIV-2 and HIV-1. EC.sub.50, .mu.g/ml
EC.sub.90, .mu.g/ml Tropism PRO 140 PA14 PRO 140 PA14 HIV-1 Isolate
CC 1/85 R5 0.14 <0.05 6.0 2.7 JR-FL R5 0.09 0.46 7.0 >100
HIV-2 Isolate A2267 R5 0.10 0.17 98 8.3 A2270 R5 0.12 3.2 3.5 4.6
310072 R5 0.08 0.07 1.4 4.4 SLRHC R5 0.57 0.35 21 14 310340 R5 4.7
7.6 >100 63 Median 0.12 0.35 21 8.2 310342 R5X4 >100 >100
>100 >100 77618 R5X4 >100 >100 >100 >100
[0326] Activity of PRO 140 and PA14 against HIV-1 reference
isolates and comparison with previous data: Two well-characterized
HIV-1 isolates, JR-FL and CC 1/85, were tested for their
susceptibility to inhibition by PRO 140. EC.sub.50 and EC.sub.90
values were determined and compared to results from prior studies
(Tables 17 and 18). All EC.sub.50 and EC.sub.90 values obtained in
the current study can be found in Tables 19-21.
TABLE-US-00015 TABLE 17 EC.sub.50 and EC.sub.90 values (ug/ml)
observed for PRO 140 against R5 HIV-1 reference viruses. Current
Study Previous Studies (n = 5) (n = 33) Virus EC50 EC90 EC50 EC90
JR-FL Median: 0.09 7.0 0.13 3.4 Mean: <0.97 22 <0.41 >10
St Dev: >1.8 36 >0.62 >15 (n = 24) CC 1/85 Median: 1.4 6.0
0.69 4.9 Mean: <7.7 23 <1.6 >12 St Dev: >16 38 >2.2
9.52
TABLE-US-00016 TABLE 18 PRO 140 EC.sub.50 and EC.sub.90 values
(ug/ml) for reference HIV-1 isolates from a previous study
(300-TD-028), where HIV-1 replication was quantitated by p24 ELISA
JR-FL CC 1/85 Source EC.sub.50 EC.sub.90 EC.sub.50 EC.sub.90
NT040805TK <0.1 1.2 0.08 0.88 NT041505TK 0.43 34 0.1 3.4
NT051005TK 0.37 3.4 1.1 13 NT071204AP-A 0.003 0.01 0.004 0.34
NT071204AP-B 0.03 18 1.4 4.2 NT071604TK-A 0.28 5.4 ND ND
NT071604TK-B 0.13 2.2 0.16 3.6 NT071604TK-C 0.05 3.1 ND ND
NT071604TK-D 0.97 12 ND ND NT071604TK-P 0.39 3.1 2.1 9.6
NT071904AP-A <0.001 0.02 0.006 0.22 NT071904AP-B <0.001
<0.001 <0.001 <0.001 NT071904AP-C <0.001 <0.001
0.001 0.01 NT071904AP-D 0.001 0.13 0.01 0.03 NT071904AP-P <0.001
0.02 <0.001 1.1 NT072304TK-A 0.03 0.52 4.5 >50 NT072304TK-B
1.8 >50 ND ND NT072304TK-C 0.24 3.1 ND ND NT072304TK-D 0.14 0.93
ND ND NT072304TK-P 0.58 5.8 0.7 4.9 NT073004TK-D 0.45 >50 0.96
>50 NT073004TK-P 0.06 8.1 0.67 1.1 NT080604TK-A 0.72 8.2 0.58
8.1 NT080604TK-C 0.13 2.1 ND ND NT080604TK-E 1.9 18 5.4 24
NT080604TK-P 0.61 10 4.6 33 NT080904AP-A 0.69 11 ND ND NT080904AP-B
0.12 2.2 ND ND NT080904AP-C 0.02 0.22 0.04 4.9 NT080904AP-P 2.7 3.7
0.85 7.3 NT081704AP-A 0.48 >50 7.8 13 NT081704AP-C 0.06 22 5.1
8.2 NT081704AP-D 0.04 18 2.7 >50 Median: 0.13 3.4 0.69 4.9 Mean:
<0.41 >10 <1.6 >12 St Dev: >0.62 >15 >2.2
>17
TABLE-US-00017 TABLE 19 PRO140 and PA14 EC (ug/ul) for HIV2 and
HIV-1 RT PRO 140 PA14 Source (pg/well) EC.sub.50 EC.sub.90 MPI
R.sup.2 EC.sub.50 EC.sub.90 MPI R.sup.2 Virus: R5 HIV-1 CC 1/85
NT111406TK 0.039 <0.046 5.971 102 0.753 <0.046 2.726 103
0.785 NT111706TK 0.018 36.943 90.136 484 0.772 27.699 87.000 210
0.794 NT120406TK 0.444 0.135 2.578 95 0.966 <0.046 1.121 100
0.990 NT121106JZ 0.251 0.054 0.199 94 0.929 <0.046 0.385 99
0.946 NT010207TK 0.131 1.667 15.539 93 0.842 0.668 13.416 107 0.901
Median: 0.131 0.135 5.971 95 0.046 2.726 103 Virus: R5 HIV-1 JR-FL
NT111406TK 0.044 <0.046 1.515 100 0.9392 <0.046 3.815 135
0.6751 NT111706TK 0.017 34.328 >100 467 0.582 32.261 >100 102
0.7984 NT120406TK 0.157 <0.046 0.499 103 0.9976 <0.046 2.300
95 0.8924 NT121106JZ 0.102 0.134 12.426 101 0.993 0.129 >100 88
0.9577 NT010207TK 0.164 3.638 75.185 110 0.7915 0.792 >100 87
0.9715 Median: 0.102 0.090 6.971 102 0.460 >100 92 Virus: R5
HIV-2 A2267 NT111406TK 0.025 0.047 0.057 97 0.9319 <0.046 0.358
98.56 0.7626 NT111706TK 0.014 4.597 98.390 115 0.7732 9.011 >100
83.12 0.799 NT120406TK 0.154 0.103 >100 88 0.7183 0.303 15.533
121.5 0.8465 NT121106JZ 0.197 <0.046 1.468 94 0.8508 <0.046
0.988 98.82 0.7686 NT010207TK 0.220 0.203 >100 75 0.992 1.431
>100 70.55 0.2136 Median: 0.154 0.103 98.390 94 0.174 8.261 99
RT: Reverse Transcriptase activity, mean of 6 wells, defined as
100% viral replication MPI: Maximum percent inhibition as
determined from the plateau of the sigmoidal curve fit by Prism
software. Values not included in Table 19 medians (Poor curve fit
(R.sup.2 < 0.7) include the first line of values for PA14/R5
HIV-1 JR-FL, the second line of values PRO140/R5 HIV-1 JR-FL, and
the fifth line of values for PA14/R5 HIV-2 A2267.
TABLE-US-00018 TABLE 20 PRO140 and PA14 EC (ug/ul) for HIV2 and
HIV-1 (continued) RT PRO 140 PA14 Source (pg/well) EC.sub.50
EC.sub.90 MPI R.sup.2 EC.sub.50 EC.sub.90 MPI R.sup.2 Virus: R5
HIV-2 A2270 NT111406TK 0.017 0.120 0.838 103 0.867 <0.046 8.021
107 0.911 NT111706TK 0.015 <0.046 3.535 97 0.939 3.326 9.832 99
0.955 NT120406TK 0.040 0.363 4.886 97 0.967 3.170 4.247 93 0.814
NT121106JZ 0.157 <0.046 0.788 103 0.739 <0.046 0.809 98 0.999
NT010207TK 0.097 0.382 9.678 101 0.971 3.531 4.614 92 0.818 Median:
0.040 0.120 3.535 101 3.170 4.614 98 Virus: R5 HIV-2 310072
NT111406TK 0.026 <0.046 <0.046 92 -- 0.078 >100 90 0.997
NT111706TK 0.032 0.576 2.057 96 0.993 2.174 7.528 97 0.956
NT120406TK 0.291 0.078 0.561 96 0.890 0.063 0.411 91 0.896
NT121106JZ 0.195 <0.046 1.360 97 0.969 <0.046 1.327 94 0.882
NT010207TK 0.166 3.103 9.664 97 0.822 3.848 4.613 95 0.676 Median:
0.166 0.078 1.360 96 0.070 4.428 92 Virus: R5 HIV-2 SLRHC
NT111406TK 0.049 <0.046 0.337 104 0.900 <0.046 0.896 101
0.737 NT111706TK 0.014 0.831 32.336 135 0.977 7.288 22.063 100
0.942 NT120406TK 0.061 0.567 14.968 127 0.836 0.351 13.730 103
0.860 NT121106JZ 0.123 0.552 21.184 150 0.861 <0.046 10.660 100
0.832 NT010207TK 0.111 4.647 40.089 228 0.965 3.688 >100 97
0.873 Median: 0.061 0.567 21.184 135 0.351 13.730 100 RT: Reverse
Transcriptase activity, mean of 6 wells, defined as 100% viral
replication MPI: Maximum percent inhibition as determined from the
plateau of the sigmoidal curve fit by Prism software. Values not
included in Table 20 medians (Poor curve fit (R.sup.2 < 0.7)
include the fifth line of values for PA14/R5 HIV-2 310072.
TABLE-US-00019 TABLE 21 PRO140 and PA14 EC (ug/ul) for HIV2 and
HIV-1 (continued) RT PRO 140 PA14 Source (pg/well) EC.sub.50
EC.sub.90 MPI R.sup.2 EC.sub.50 EC.sub.90 MPI R.sup.2 Virus: R5
HIV-2 310340 NT111406TK 0.051 <0.046 <0.046 87 0.202
<0.046 <0.046 -- -- NT111706TK 0.024 13.862 >100 90 0.941
15.137 >100 181 0.876 NT120406TK 0.135 0.053 >100 78 0.822
0.114 1.641 179 0.559 NT121106JZ 0.663 0.052 >100 87 0.806
<0.046 26.304 107 0.873 NT010207TK 0.278 9.437 >100 74 0.861
12.121 >100 83 0.515 Median: 0.135 4.745 >100 83 7.591 63.152
144 Virus: R5X4 HIV-2 310342 NT111406TK 0.230 >100 >100 NA --
>100 >100 NA -- NT111706TK 0.018 >100 >100 NA --
>100 >100 NA -- NT120406TK 0.114 >100 >100 NA --
>100 >100 14 -- NT121106JZ 0.793 >100 >100 NA --
>100 >100 NA -- NT010207TK 0.096 >100 >100 NA --
>100 >100 -21 -- Median: 0.114 >100 >100 -- >100
>100 -4 Virus: R5X4 HIV-2 77618 NT111406TK 0.023 >100 >100
6 -- >100 >100 4 -- NT111706TK 0.017 >100 >100 NA --
>100 >100 NA -- NT120406TK 0.129 >100 >100 NA --
>100 >100 NA -- NT121106JZ 0.203 >100 >100 -2 --
>100 >100 -20 -- NT010207TK 0.072 >100 >100 NA --
>100 >100 -16 -- Median: 0.072 >100 >100 2 >100
>100 -16 Median Values, all R5 HIV-2 Viruses 0.120 21.184 96 --
0.351 8.261 99 -- RT: Reverse Transcriptase activity, mean of 6
wells, defined as 100% viral replication MPI: Maximum percent
inhibition as determined from the plateau of the sigmoidal curve
fit by Prism software. Values not included in Table 21 medians
(Poor curve fit (R.sup.2 < 0.7) include the first line of values
for PRO140/R5 HIV-2 310340, and first, third and fifth line of
values for PA14/R5 HIV-2 310340. NA: Not applicable; Prism could
not fit a sigmoidal curve.
[0327] The EC values were measured by p24 ELISA in previous
studies, according to published methods. Because the p24 ELISA
cannot efficiently quantitate p24 from the HIV-2 isolates used in
this study, a commercial ELISA that measures reverse transcriptase
was chosen as a readout for both the HIV-2 and HIV-1 infection
assays in the present study. As indicated in Table 17, comparable
results were observed using the two assay methods.
Susceptibility of HIV-2 Isolates to PRO 140 and PA 14:
[0328] The susceptibility of five R5 HIV-2 isolates to PRO 140 and
PA14 was determined alongside the two HIV-1 reference isolates and
two R5X4 HIV-2 isolates as controls. The panel was tested five
times on a different PBMC donor pool each time, using the RT ELISA
as a readout. The median EC.sub.50 values are shown in FIG. 21.
Full EC.sub.50 and EC.sub.90 data can be found in Tables 19-21
above. Against the R5 HIV-2 isolates, PRO 140 had a median
EC.sub.50 of 0.12 ug/ml, a median EC.sub.90 of 21 ug/ml, and a
median MPI of 96% (FIGS. 22A-C). These results are similar to those
observed for the two R5 HIV-1 reference isolates that were tested
in parallel. PRO 140 did not measurably inhibit replication of R5X4
HIV-2 isolates in this study. PRO 140 and PA14 demonstrated similar
antiviral activities.
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